Methods for treating covid-19

ABSTRACT

The invention relates to methods for treating COVID-19 by targeting the inflammasome/caspase1/pyroptosis axis as a key inflammatory pathway. In particular, the invention relates to treating a patient infected with SARS-CoV-2 with an effective amount of one or more compounds that directly or indirectly inhibit one or more pathways of the inflammasome/caspase1/pyroptosis axis.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of International ApplicationNo. PCT/US2021/030063 filed Apr. 30, 2021 and which published asInternational Publication No. WO 2021/222687 on Nov. 4, 2021, and whichclaims benefit of and priority to US provisional patent applicationSerial Nos. 63/018,923 filed 1 May 2020, 63/156,479 filed 4 Mar. 2021and 63/161,635 filed 16 Mar. 2021.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said ASCII copy was created Jun. 30, 2021, is namedF7259-00036_SL.txt and is 525 bytes in size.

FIELD OF THE INVENTION

The invention relates to methods for treating COVID-19 by targeting theinflammasome/caspase1/pyroptosis axis as a key inflammatory pathway.

BACKGROUND OF THE INVENTION

Beginning in December 2019, a novel coronavirus first detected in Wuhan,China, and designated SARS-CoV-2, has caused an international outbreakof respiratory illness termed Covid-19. Since the initial detection ofthe virus, millions of cases of Covid-19 have been confirmed worldwide,with the first reported cases in the US occurring on Jan. 19, 2020(Holshue et al. N Engl J Med. 2020; 382(10): 929-936). The full spectrumof Covid-19 ranges from mild, self-limiting respiratory tract illness tosevere progressive pneumonia, multiorgan failure, and death (Chen et al.Lancet. 2020; 395(10223): 507-513). Initial reports, first from Chinaand then from Italy, stressed high mortality rates, ranging from 2.3%overall to as high as 15% in those aged 80 years and older (Grasselli G,A. Pesenti A, and M. Cecconi M. Critical Care Utilization for theCOVID-19 Outbreak in Lombardy, Italy: Early Experience and ForecastDuring an Emergency Response. JAMA. 2020). However, this is likely anoverestimate since the prevalence of asymptomatic carriers is as yetunknown. What is increasingly clear, though, is that individuals athighest risk are those over 65 years with concomitant chronic conditionswith an inflammatory profile such as diabetes, pre-diabetes, elevatedBMI, liver disease, and chronic renal impairment.

After the emergence of severe acute respiratory syndrome (SARS) in 2003,screening of approved drugs identified lopinavir, a humanimmunodeficiency virus (HIV) type 1 aspartate protease inhibitor, ashaving in vitro inhibitory activity against SARS-CoV, the virus thatcauses SARS in humans (Chen et al. J Clin Virol. 2004; 31(1): 69-75; Chuet al. Thorax. 2004; 59(3): 252-256; Wu et al. Proc Natl Acad Sci USA.2004; 101(27): 10012-10017). Ritonavir is combined with lopinavir toincrease its plasma half-life through the inhibition of cytochrome P450.An open-label study published in 2004 suggested, by comparison with ahistorical control group that received only ribavirin, that the additionof lopinavir—ritonavir (400 mg and 100 mg, respectively) to ribavirinreduced the risk of adverse clinical outcomes (acute respiratorydistress syndrome or death) as well as viral load among patients withSARS (Chu et al. Thorax. 2004; 59(3): 252-256). However, the lack ofrandomization and the concomitant use of glucocorticoids and ribavirinin that study made the effect of lopinavir-ritonavir difficult toassess.

Recently, the combined effect of lopinavir-ritonavir in the treatment ofSARS-CoV-2 was tested in a randomized study involving 200 patients inChina. Unfortunately, there was no improvement in clinical symptoms,mortality, or viral loads in the lopinavir-ritanovir group compared tostandard of care alone (Chu et al. Thorax. 2004; 59(3): 252-256).Hydroxychloroquine, an anti-malarial agent, combined with azithromycin,an anti-bacterial agent, were recently used in 22 patients withSARS-CoVid-2 infection in France. Viral loads appeared to diminishfaster in the treated patients than in the 2 control patients, but noconclusions can be drawn from such small numbers and anyway no mentionwas made of its impact on clinical symptoms.

The role of the inflammasome/caspase1/pyroptosis axis as a keyinflammatory pathway in the context of coronavirus infections and beyondis well known in the art. (files labelled CoV-01-CoV-06).

Yue et al. (Cell Death and Disease (2018) 9:904) found that ReceptorInteracting Protein 3 (Rip3)-mediated oligomerization of SARS 3a (thelargest of the SARS-CoV accessory protein open reading frames) causesnecrotic cell death, lysosomal damage, and caspase-1 activation—alllikely contributing to the clinical manifestations of SARS-CoVinfection. Yue et al. also found that SARS 3a activates caspase-1 eitherdirectly or via an enhanced potassium efflux, which triggers NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasome assembly.

Shi et al. (Cell Death Discovery (2019) 5:101) identified severalmechanisms by which a SARS-CoV open reading frame (ORF) activatesintracellular stress pathways and targets the innate immune response byactivating NLRP3 inflammasomes.

Zalinger et al. (J. Neurovirol. (2017) 23:845-854) used murinecoronavirus, mouse hepatitis virus (MHV), infection of the centralnervous system and liver to assess of the role of the inflammasome andits related cytokines on pathogenesis and host defense during viralinfection. Their data suggest that inflammasome signaling is largelyprotective during murine coronavirus infection, in large part due to thepro-inflammatory effects of IL-18.

Jiang et al. (Viruses 2019, 11, 39; doi:10.3390/v11010039) found thatMERS-CoV infection induced pyroptosis and over-activation of complementin human macrophages. Jiang et al.'s data indicate that MERS-CoVinfection induces overactivation of complement, which may contribute topyroptosis and inflammation. Pyroptosis and inflammation were suppressedby inhibiting C5aR1.

Kesavardhana et al. (Annu. Rev. Immunol. 2020. 38:567-95) reviewedmechanisms governing caspase activation and programmed cell death withspecial emphasis on the recent progress in caspase cross talk andcaspase-driven gasdermin D (GSDMD)-induced pyroptosis.

Guo et al. (PLoS Pathog 2015 11(9): e1005155.doi:10.1371/journal.ppat.1005155) demonstrate that the reactive oxygenspecies (ROS)/nucleotide-binding domain-like receptor protein 3(NLRP3)/interleukin-1β (IL-1β) axis institutes an essential signalingpathway, which is over activated and directly causes the severe liverdisease during viral infection, which sheds light on development ofefficient treatments for human viral fulminant hepatitis and othersevere inflammatory diseases.

The relevance of the inflammasome/caspase1/pyroptosis axis specificallyin COVID-19 is also known in the art.

Parisi and Leosco (Precision medicine in COVID-19: IL-1β a potentialtarget, JACC: Basic to Translational Science (2020)) speculated about i)the role of NLRP3 in the clinical variability of COVID-19; ii) thepotential therapeutic effect in COVID-19 of IL-1β inhibition(canakinumab, anakinra), and iii) the role of visceral adipose tissue inthe inflammatory response to SARS-CoV-2 infection.

Conti et al. (Journal of biological regulators and homeostatic agentsVolume 32 Issue 2 2020) speculated about anti-inflammatory strategies,specifically the induction of pro-inflammatory cytokines (IL-1 and IL-6)and lung inflammation by Coronavirus-19 (COVID-19 or SARS-CoV-2).

Yang (Cell Pyroptosis, a Potential Pathogenic Mechanism of 2019-nCoVInfection (Jan. 29, 2020). Available at SSRN:https://ssrn.com/abstract=3527420 orhttp://dx.doi.org/10.2139/ssrn.3527420) discussed the relationshipbetween 2019-nCoV infection and cell pyroptosis. Yang's hypothesis ofthe relationship between 2019-nCoV and cell pyroptosis is presented inFIG. 1 .

Deftereos et al. (Hellenic Journal of Cardiology, 2020,https://doi.org/10.1016/j.hjc.2020.03.002) asked whether colchicine,administered in a relatively low dose, could potentially have an effecton the patients' clinical course by limiting the myocardial necrosis andpneumonia development in the context of COVID-19. If present, thiseffect would be attributed to its potential to inhibit inflammasome and(less probably) to the process of SARS-CoV-2 endocytosis in myocardialand endothelial respiratory cells.

There is a need for effective therapeutic agents to treat coronavirusinfections, in particular SARS-CoV-2.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention relates to methods for treating COVID-19 by targeting theinflammasome/caspase1/pyroptosis axis as a key inflammatory pathway. Inparticular, the invention relates to treating a patient infected withSARS-CoV-2 or a variant thereof with an effective amount of one or morecompounds that inhibit one or more pathways of theinflammasome/caspase1/pyroptosis axis. Compounds that inhibit one ormore pathways of the inflammasome/caspase1/pyroptosis axis includecompounds that directly or indirectly inhibit the one or more pathwaysas direct and indirect inhibitors of pathways that are upstream ordownstream of the inflammasome/caspase1/pyroptosis axis.

The present invention relates to methods for treating a patient infectedwith SARS-CoV-2 or a variant thereof and having pyroptotic activitywhich may comprise (a) determining if the patient is infected withSARS-CoV-2 or a variant thereof, (b) optionally determining if thepatient has pyroptotic activity, and (c) administering an effectiveamount of a compound that inhibits one or more pathways of theinflammasome/caspase1/pyroptosis axis to the patient infected withSARS-CoV-2 or a variant thereof.

In another embodiment, the invention relates to screening a markerindicative of pyroptotic activity, wherein the marker has increasedactivity if pyroptotic activity is present as compared to the expressionof the marker in the absence of pyroptotic activity. In an advantageousembodiment, the marker may be NOD-, LRR- and pyrin domain-containingprotein 3 (NLRP3), IL-1 receptor antagonist (IL-1RA), interleukin-1β(IL-1β), interleukin-18 (IL-18), gasdermin D (GSDMD), and/or caspase 1.

In an advantageous embodiment, the compound that inhibits one or morepathways of the inflammasome/caspase1/pyroptosis axis is a caspase 1inhibitor.

In a particularly advantageous embodiment, an effective amount of thecompound that inhibits one or more pathways of theinflammasome/caspase1/pyroptosis axis may be about 10 mg/kg to about 100mg/kg or about 300 mg to about 900 mg per dose or about 300 mg to about3600 mg per day. Advantageously, the effective amount may be about 600mg three times a day to about 900 mg three times a day. Advantageously,the administering is oral.

The methods of the invention may further comprise administering aneffective amount of a caspase 1, GSDMD, IL-1R, IL-1β, IL-18, or NLRP3inhibitor.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U. S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 : A hypothesis of the relationship between 2019-nCoV and cellpyroptosis set forth in Yang (Cell Pyroptosis, a Potential PathogenicMechanism of 2019-nCoV Infection (Jan. 29, 2020). Available at SSRN:https://ssrn.com/abstract=3527420 orhttp://dx.doi.org/10.2139/ssrn.3527420).

FIG. 2A: Clinical Course of an Immunosuppressed Patient with RapidDeterioration Due to COVID-19. Chronology of patient clinical course,treatment regimen, absolute blood counts, clinical immunomonitoringdata, and inflammatory markers. Red shading denotes that a given valueis outside the reference range. Profound CD4 and CD8 T-cell lymphopeniais detected on day 4 after onset of symptoms (d4). On day 7 (d7),despite CD8 T-cell counts normalizing, CD4 Tcells remain in thelymphopenic range, despite an uptrend. NK and B-cell absolute counts andT-cell activation markers CD25 and HLA-DR are normal. Dendritic cellsare absent in peripheral blood.

FIG. 2B. Images of patient chest x-rays and non-contrast chest CT scan.On day 2 after onset of symptoms (d2), the chest x-ray showed bilateralperihilar and basilar predominant airspace opacities, which were againdemonstrated on day 4. On day 7 after onset of symptoms (d7), the CTscan showed diffuse bilateral pulmonary opacities characterized bysevere groundglass opacities and moderate dependent consolidations.

FIG. 3A: Immunological Phenotype of an Immunosuppressed Patient withRapid Deterioration Due to COVID-19 based on clinical immune phenotypingof peripheral blood samples obtained day 7 after onset of COVID-19symptoms (d7). Innate Immune Phenotype: CD16+ monocytes showedsignificant upregulation of CD11b and proinflammatory CD14+CD16+monocytes showed upregulation of CD38. Approximately 15% of B-cellsshowed surface tetherin staining. Monocyte cytoplasmic IL6, MIP1α,MIP1β, and TNF-α levels were overall normal compared to controls (datanot shown).

FIG. 3B. T Cell Phenotype: There was a decrease inCXCR5+ICOS+T-follicular helper cells and complete absence ofCD127-CD25+T-reg cells. The majority of CD4 T-cells were naïve andphosphorylated STAT-4 late and poorly on IL-12 stimulation. CD8 T-cellsshowed strong upregulation of CD38.

FIG. 3C. Cytotoxic Immune Phenotype: CD8 T-cells showed decrease in % ofperforin positive cells. No changes were observed for NK-cellperforin/granzyme staining.

FIG. 3D. Humoral Immune Phenotype: B-cells showed profound loss of CD27+memory, as well as surface IgG and IgA. SARS-CoV-2-specific IgG and IgMwere absent (see also FIG. 2A).

FIG. 3E Pyroptosis: There was increase in caspase-1 staining ofCD45+CD3+ T-cells as well as CD45+CD3− cells. Caspase-3 staining was notdifferent than controls (data not shown).

FIG. 4 : The Inflammasome and COVID-19: Rational for belnacasan.

FIGS. 5A-5C: Subject Diary.

FIG. 6 : Schedule of Events. ¹Screening tests: HIV, Hepatitis B,Hepatitis C, and pregnancy (serum pregnancy test for women ofchild-bearing potential per MedStar definition at enrollment and at day14). ²Vital signs: oral temperature, heart rate, systolic bloodpressure. ³Quantitative nasopharyngeal SARS-CoV-2 RNA testing (RT-PCR):nasopharyngeal or throat swabs are used; any FDA-approved or commercialtesting method is acceptable. ⁴Standard labs: serum chemistry,coagulation tests and hematology. ⁵Blood draws: serum and peripheralblood mononuclear cells for later immunological analysis. ⁶Immunologytests: COVID-19 relevant immunology labs. ⁷Quantitative blood SARS-CoV-2antibody testing: Blood samples are used; any FDA-approved or commercialtesting method is acceptable. ⁸WHO 9-point scale: see description ofsecondary end points for details. ⁹Day 1 (i.e., the first day of drugintake) is most likely also the screen/enroll day; subjects receive aphone call from Study Team a few hours after first dose is taken tocheck up on how they are doing. ¹⁰ET: Subjects who prematurelydiscontinue study drug for any reason are asked to have an EarlyTermination (ET) visit the day the last study drug dose was taken.

DETAILED DESCRIPTION OF THE INVENTION

The role of the inflammasome/caspase1/pyroptosis axis as a keyinflammatory pathway in the context of coronavirus infections and beyondis well known in the art. Furthermore, the relevance of theinflammasome/caspase1/pyroptosis axis specifically in COVID-19 is alsoknown in the art. Therefore, clinical data that provides a nexus betweenthe inflammasome/caspase1/pyroptosis axis and COVID-19 provides insightfor treating COVID-19 by targeting pathways in theinflammasome/caspase1/pyroptosis axis.

The liver disease and transplant communities provide a window intopopulations that are especially important to study in light ofcoronavirus disease 2019 (COVID-19) as these patients typically havemany co-morbidities, including diabetes, obesity and related cardiacissues, cancer, and most notably end-stage organ failure. What theseco-morbidities have in common is underlying chronic inflammationinvolving activation of the inflammasome (Guo H, Callaway J B, Ting J P.Inflammasomes: mechanism of action, role in disease, and therapeutics.Nat Med 2015; 21:677-687). What these patients have in common is lethalexperiences with COVID-19 (Onder G, Rezza G, Brusaferro S. Case-FatalityRate and Characteristics of Patients Dying in Relation to COVID-19 inItaly. JAMA 2020). Putting these two factors together caused Applicantsto speculate that the inflammasome, and more specifically pyroptosis,are what may drive tragic outcomes for patients with co-morbidities inthe face of COVID-19. Applicants observed this phenomenon in a livertransplant patient with several co-morbidities, who rapidly succumbed tothe disease, and whose clinical course and immunological phenotype aredescribed in Example 1. Applicants' finding has been corroborated withat least ten other patients.

Applicants' findings shed new light on why and how SARS-CoV-2 infectionscause such rapid lethality. T cell lymphopenia and lack of COVIDspecific IgM indicates that the adaptive immune system never properlykicked in. Caspase-1 overexpression in lymphocytes suggests pyroptosisas one of the possible mechanisms of T-cell depletion and functionalabnormalities of other lymphocytes (Doitsh et al. Nature 2014;505:509-514).

Applicants propose that the inflammatory response is secondary to thedanger signals from pyroptotic cell death of immune system cells,resulting in a heightened inflammation compared to the one induced byapoptotic tissue cells. The end result is likely a self-damaging shutdown of the immune system that further fuels the inflammation created bythe viral infection. Applicants provide data backed connection betweencaspase-1 and COVID-19, a connection with significant therapeuticimplications as rather than inhibition of the inflammatory response,preventing the pyroptotic lymphocyte death fueling the inflammationbecomes more critical.

Based upon knowledge of the inflammasome/caspase1/pyroptosis axis as akey inflammatory pathway in the context of coronavirus infections andthe hypotheses regarding COVID-19, clinical data showing inflammasomeactivation, and more specifically increased levels of lactatedehydrogenase (LDH) Rayamajhi et al. Methods Mol Biol 2013; 1040:85-90and overexpression of caspase-1 and pyroptosis, confirms the hypothesisset forth in FIG. 1 .

By targeting key components of the inflammasome/caspase1/pyroptosis axisto minimize or prevent pyroptosis, Applicants believe the onset and/orseverity of lympophenia, hyperinflammation of other immune cells includemonocytes and subsequently COVID-19 is delayed and/or mitigated andpossibly prevented.

Rodrigues et al. (J. Exp. Med. 2020 Vol. 218 No. 3https://rupress.org/jem/article/218/3/e20201707/211560/Inflammasomes-are-activated-in-response-to-SARS)found that the NLRP3 inflammasome is activated in response to SARS-CoV-2infection and is active in COVID-19 patients by studying moderate andsevere COVID-19 patients, they found active NLRP3 inflammasome in PBMCsand tissues of postmortem patients upon autopsy. Inflammasome-derivedproducts such as Casp1p20 and IL-18 in the sera correlated with themarkers of COVID-19 severity, including IL-6 and LDH. Moreover, higherlevels of IL-18 and Casp1p20 are associated with disease severity andpoor clinical outcome. Their results suggest that inflammasomesparticipate in the pathophysiology of the disease, indicating that theseplatforms might be a marker of disease severity and a potentialtherapeutic target for COVID-19.

Todo et al. (Inflammation Research (2021) 70:7-10https://doi.org/10.1007/s00011-020-01413-2) also identified the presenceof NLRP3 inflammasome aggregates in the lungs of fatal COVID-19pneumonia thus providing the potential molecular link between viralinfection and cytokine release syndrome.

Sars-CoV-2-induced caspase1 activation and pyroptosis is confirmed inmonocytes, which may be as important as in lymphocytes (see, e.g.,https://www.nature.com/articles/s41420-021-00428-w.pdf). There is notonly upregulation of caspase1 in SARS-CoV-2 infected monocytes but alsoeffective inhibition of resulting pyroptosis via a caspase1 inhibitor.

Potential inhibitors of NLRP3 inflammasome and their targets are setforth in the below table (see Zahid et al. (2019) Front. Immunol.10:2538 doi: 10.3389/fimmu.2019.02538), all of which are contemplated aspharmaceutical compounds for treating COVID-19 in the present invention.

Agent Target(s) Potential mechanism Glyburide NLRP3 (indirectly)Inhibits ATP-sensitive K⁺ channels; downstream of P2X7 resulting ininhibition of ASC aggregation 16673-34-0 NLRP3 (indirectly) InducesNLRP3 conformational changes secondary to its activation or binding toASC JC124 NLRP3? Blocks the expression of NLRP3, ASC, caspase-1,pro-IL-1β, TNFα and iNOS FC11A-2 NLRP3 (indirectly) Interferes withproximity induced autocleavage of pro-caspase-1, suppresses IL- 1β/18release Parthenolide NLRP1, NLRP3 Alkylates cysteine residues incaspase-1 and inflammasome, Caspase-1, NF- in ATPase domain of NLRP3,inhibits κB, IKKβ kinase activity NLRP3 ATPase activity VX-740 Caspase-1Covalent modification of the catalytic cysteine residue in the activesite of caspase-1 resulting in caspase-1 blocking and resultant cleavageof pro-IL-1β/18 VX-765 Caspase-1 Covalent modification of the catalyticcysteine residue in the active site of caspase-1 resulting in caspase-1blocking and resultant cleavage of pro-IL-1β/18 Bay 11-7082 NLRP3, IKK,E2/3 enzymes, Alkylates the cysteines in the ATPase domain PTPs ofNLRP3, inhibits NLRP3 ATPase activity BHB NLRP3 (Indirectly) Inhibits K⁺efflux resulting in reduced oligomerization of ASC and IL-1β/18 releaseMCC950 NLRP3 Blocks the ATPase domain of NLRP3 resulting in inhibitionof canonical and non- canonical NLRP3 inflammasome activation MNS NLRP3Inhibits NLRP3 ATPase activity by cysteine modification, blocks NLRP3inflammasome activation CY-09 NLRP3 Inhibits NLRP3 ATPase activity,blocks NLRP3 inflammasome activation Tranilast NLRP3 Binds to NLRP3NACHT domain to block NLRP3-NLRP3 and NLRP3-ASC interaction OLT1177NLRP3 Inhibits NLRP3 ATPase activity, blocks NLRP3 inflammasomeactivation Oridonin NLRP3 Binds to cysteine 279 of NLRP3 to abolishNLRP3-NEK7 interaction, blocks NLRP3 inflammasome activation

The present invention relates to determining if a patient is infectedwith SARS-CoV-2 and whether there is pyroptotic activity in the cells ofthe patient. For determining infection, a molecular or viral test,usually RT-PCR, is preferred over a serological or antibody test. Fordetermining pyroptotic activity, markers such as NLRP3, IL-1δ, gasderminD (GSDMD) and/or caspase 1 are screened at either the nucleic acid orprotein level wherein elevated levels of any of the markers areindicative of pyroptosis.

If a patient tests positive for COVID-19, then the patient is treatedwith an effective amount of one or more compounds that inhibit one ormore pathways of the inflammasome/caspase 1/pyroptosis axis.

Caspase-1 has been identified to be a key enzyme in theinflammasome/caspase1/pyroptosis axis because caspase-1 both activatesgasdermin and activates IL-1β and IL-18. Caspase-1 is also known as theinterleukin-converting enzyme (ICE).

Prospective clinical applications of caspase inhibitors are set forth inthe below table (see Kudelova et al., JPP No 4/2015 article 01http://jpp.krakow.pl/journal/archive/08_15/articles/01_article.html)

STRUCTURE AND DRUGS SUBSTRATE CLINICAL TRIALS company SPECIFITYPRECLINICAL DATA CLINICAL TRIALS RUNNING/TERMINATED Pralnacasan Prodrugof orally Collagenase-induced OA (27) Rheumatoid arthritis andTerminated in Phase IIb in RA due to VX-740 active reversible Type IIcollagen-induced osteoarthritis (significant liver toxicity in animals(102) Vertex peptidomimetic arthritis in mice amelioration of RA, butnot Pharmaceuticals casp-1 inhibitor Dextran sulfate sodium- OA) inducedcolitis in mice (101) Belnacasan Prodrug of orally Mouse models of acuteCompleted Phase II in Any results released VX-765 active reversibleseizures and chronic epilepsy psoriasis Vertex peptidomimetic (134)Completed Phase II in Terminated Phase IIb due to lack ofPharmaceuticals casp-1 inhibitor Dermatitis model resistant partialepilepsy efficacy Arthritis model (103) Evaluated the efficacy andPrevents CD4 T-cell safety Phase IIb in resistant pyroptotic death in adose- partial epilepsy dependent manner in HIV- infected lymphoidtissues (136) Nivocasan Orally active Fibrosis/apoptosis animalCompleted Phase II in Reductions in ALT levels in NASH GS-9450irreversible models NASH patients (6) Gilead Sciences peptidomimeticBleomycin induced Completed Phase IIa in Terminated Phase IIb due tocasp-1, -8, -9 pulmonary fibrosis in mice chronic HCV significantlaboratory abnormalities inhibitor (132) Evaluated the efficacy and andadverse events in a number of safety Phase II in chronic clinical studyparticipants HCV for 6 months Emricasan Orally active broad α-F as modelof apoptotic Completed Phase II in Efficacy in post liver transplant forIDN-6556 spectrum hepatitis in mice treatment HCV chronic HCV (108, 109)Conatus irreversible Bile duct ligated model of Pharmacokinetics andPharmaceuticals peptidomimetic liver failure, injury, fibrosis inpharmacodynamics Phase II in ACLF caspase inhibitor mice (133) (111)Post transplantation-liver, Completed Phase II of Efficacy and safetyPhase II in islets (7) safety and efficacy in NAFLD and raisedtransaminases Models of NASH, NAFLD patients undergoing liver (133, 135)transplantation Terminated Phase II in severe AH and contraindicationsto steroid therapy due to inadequate dose of Emricasan Safety Phase I/IIin islet transplantation in type I diabetic participants (112)Pharmacokinetics and pharmacodynamics Phase I in subjects with severerenal impairment and matched healthy volunteers Pharmacokinetics andpharmacodynamics Phase I in subjects with hepatic impairment and matchedhealthy volunteers

The present invention particularly relates to a caspase 1/interleukin-1βconverting enzyme (ICE) inhibitor in its prodrug form (VX-765), alsoknown as RVT-201, or in its active form (see, e.g., WO01/90063, U.S.Pat. Nos. 7,417,029, 8,329,662, 9,156,880, 9,487,555, 9,994,613, andUS20200048306, the disclosures of which are incorporated by reference)in treating COVID-19. In addition to COVID-19, the compound andpharmaceutical compositions thereof are useful as agents to treatinterleukin-1-(IL-1), apoptosis-, other forms of cell death such aspyroptotic cell death and necrotic cell death, interferon-6 (IL-6),interferon-17 (IL-17), interferon-18-(IL-18), interferon-γ (IFNγ) ortumor necrosis factor-α (TNFα) mediated disease, including inflammatorydiseases, autoimmune diseases, destructive bone disorders, proliferativedisorders, infectious diseases, and degenerative diseases. Thisinvention also relates to methods for inhibiting caspase 1/ICE activityand decreasing IL-18 production and IFN-γ production and methods fortreating interleukin-1, apoptosis-, and interferon-γ-mediated diseasesusing the compositions of this invention. The compound is represented byformula I (VX-765):

Compound I (VX-765) may be used alone or in combination with othertherapeutic or prophylactic agents, such as antibiotics, antivirals,immunomodulators, or other anti-inflammatory agents, for the treatmentor prevention of diseases mediated by NLRP3, IL-1, pyroptosis,apoptosis, IL-6, IL-18, IFNγ, or TNF-α. This invention also relates topharmaceutically acceptable derivatives and prodrugs of the compound.

Compound I (VX-765) itself is a prodrug that undergoes bioconversion toan active caspase 1/ICE inhibitor II:

Compound I (VX-765) has better in vivo activity upon oral and/orintravenous administration than the parent or active form of the drug.The active form, aspartic aldehyde II, exhibits less than optimal invivo activity, primarily because of poor bioavailability, and istherefore not well-suited for direct therapeutic use.

VX-765 is advantageously formulated as a solution or a tablet (see,e.g., Wannamaker et al., WET 321:509-516, 2007; Stack et al., J. Immunol205; 175:2630-2634 and Bialer et al., Epilepsy Research (2013) 103,2-30). The dosage of VX-765 may be about 10 to 100 mg/kg, such as about10 mg/kg, about 12.5 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75mg/kg or about 100 mg/kg. If formulated as a liquid, VX-765 is dissolvedin about 20% to about 25% Kolliphor® or any other suitablepolyethoxylated castor oil. A tablet of VX-765 may be about 300 mg pertablet with daily dosage of about 900 mg to about 3600 mg. The abovedosages may be extrapolated to other pharmaceutical compositions of thepresent invention.

In another embodiment, VX-740 (pralnacasan) is also contemplated for thepresent invention. Pralnacasan is an orally bioavailable pro-drug of apotent, non-peptide inhibitor of caspase 1/ICE having the structure:

Other caspase 1/ICE inhibitors are contemplated for the presentinvention, such as Conatus' lead caspase 1 inhibitor CTS-2090.

Additional caspase 1/ICE inhibitors contemplated for the presentinvention are presented below and set forth in WO 2017/079566:

In another embodiment, an IL-1β blocking agent may be administered byitself or in combination with a caspase 1/ICE inhibitor. Examples ofIL-1β blocking agents include, but are not limited to, Canakinumab(Ilaris®), Anakinra (Kineret®) and Rilonacept (Arcalyst®).

In yet another embodiment, the pharmaceutical composition of the presentinvention may be administered in combination with another therapeuticagent, advantageously one used for COVID-19. Examples of such candidatedrug treatments in Phase III-IV trials are presented in the below table.

Existing disease Trial Expected Drug candidate Description approvalsponsor(s) Location(s) results Remdesivir antiviral; adenosineinvestigational^([77]) Gilead, WHO, China, Japan initially; Aprilnucleotide analog INSERM expanded internationally (Chinese, inhibitingRNA in Global Solidarity and Japanese synthesis in Discovery Trialstrials) to coronaviruses mid-2020 Hydroxychloroquine antiparasitic andmalaria, rheumatoid CEPI, WHO, Multiple sites in China; April 2020 orchloroquine antirheumatic; generic arthritis, lupus INSERM GlobalSolidarity and (Chinese made by many (International)^([83][84])Discovery Trials, trials); mid- manufacturers Europe, international 2020Favipiravir antiviral against influenza (China)^([86]) Fujifilm ChinaApril 2020 influenza Lopinavir/ritonavir antiviral, immuneinvestigational CEPI, WHO, Global Solidarity and mid-2020 without orwith suppression combination; UK Discovery Trials, Rebiflopinavir/ritonavir Government, multiple countries approved^([88]) Univ,of Oxford, INSERM Sarilumab human monoclonal rheumatoid arthritisRegeneron- Multiple countries Spring 2020 antibody against (USA,Europe)^([89]) Sanofi interleukin-6 receptor ASC-09 + ritonavirantiviral combination not Ascletis Multiple sites in China Spring 2020approved; ritonavir Pharma approved for HIV^([88]) Tocilizumab humanmonoclonal immunosuppression, Genentech- Multiple countries mid-2020antibody against rheumatoid arthritis Hoffmann-La interleukin-6 receptor(USA, Europe)^([92]) Roche Dapagliflozin sodium-glucose hypoglycemiaSaint Luke's Multiple countries December cotransporter 2 agent^([94])Mid America 2020 inhibitor Heart Institute, AstraZeneca CD24Fc antiviralnew drug candidate Oncolmmune, Multiple sites in the 2021immunomodulator Inc. United States against inflammatory response

Other Drugs used for COVID (see, e.g., Yu Peng, Hongxun Tao, SenthilKumaran Satyanarayanan, Kunlin Jin, Huanxing Su. A Comprehensive Summaryof the Knowledge on COVID-19 Treatment Aging Dis. 2021 February; 12(1):155-191. Published online 2021 Feb. 1. doi: 10.14336/AD.2020.1124.PMCID: PMC7801274 and Shagufta, Irshad Ahmad. The race to treatCOVID-19: Potential therapeutic agents for the prevention and treatmentof SARS-CoV-2. Eur J Med Chem. 2021 Mar. 5; 213: 113157. Publishedonline 2021 Jan. 12. doi: 10.1016/j.ejmech.2021.113157. PMCID:PMC7802596). Apart from the direct action of the SARS-Cov 2 viruscytokines have a direct role in the immunopathogenesis of COVID-19 byinducing hyper inflammation and lung injury that defines this diseaseand the severe phenotype. Monocytes and macrophages have an importantrole in respiratory failure during COVID-19; these cells migrate to thelungs, producing pro-inflammatory cytokines, and inducing epithelialdamage. COVID-19 patients present an impaired immune response due toexhausted phenotype and lower effector T cells, CD8+T lymphocytes, andNK cells, culminating in antiviral immunity loss. While vaccines are nowavailable a proportion of individuals continue to develop a severephenotype, either from vaccine failure or not being vaccinated. Thefollowing are a guide to potential and actual therapies that maymitigate the effects of the virus directly or by reducing theinflammatory consequences.

Vitamin supplementation enhances immune response: Vitamin A, D, E and C,selenium and zinc. The above supplements have a variety of effectsincluding immune modulation, particularly vitamin D, improving recoveryfrom viral infections, vitamins A and E and zinc and an antioxidanteffect selenium and vitamin C. They may have a role in immune“exhaustion”.

Polyclonal (pAbs) and monoclonal (mAbs) antibody-based immunotherapy.Monoclonal Abs are primarily manufactured however pAbs are from pooledhuman blood products also known as convalescent plasma (CP) or immuneplasma. Antibodies prevent the virus from entering the host cells,blocking receptor ACE2 and directly on the virus (neutralizingantibodies [nAbs] recognize epitopic regions of SARS-CoV-2). On thevirus, they prevent its infectivity by activating several pathways, suchas the complement system, cell cytotoxicity, and phagocytic clearance.The therapeutic use of mAbs extends to the use of many that block orotherwise modulate part of the antibody cascade. These include anti-JAK,anti-GM-CSF, anti-GM-CSF receptor, anti-M-CSF receptor, anti-CD14,anti-IFN?, anti-VEGF, anti-BKT, anti-CCR5, anti-IL-6, anti-IL-6receptor, anti-TNFa, anti-IL1ß, anti-IL1ß receptor, and complement C5inhibitor. Some antibody drugs are being used to disrupt criticalcomplication that increase morbiditiy and include the hyper-thromboticstate found in critically ill patients by using anti-P-selectin,anti-CTGF, and factor XIIa antagonist mAbs. to restore the exhausted Tlymphocytes' and NK cells' immunity, anti-PD1 mAbs. Another therapeuticstrategy using antibodies is intravenous immunoglobulin (IVIg) thatcontains polyclonal IgG isolated from healthy donors, which can befurther enhanced by using IgG antibodies collected from recoveredCOVID-19 patients in the same geographical region as the patient.

Immune cell-based therapy: NK and T cells including CART, stem cellsfrom any source and related products. A viable approach is the use ofallogeneic human leukocyte antigen-matched umbilical cord-derived Tregs(UBC-Treg) which can be widely expanded and used on a larger scale.

Cytokine use in relation T-cells includes IL-7 to increase CD4+ andCD8+T lymphocyte counts without inducing the production ofpro-inflammatory mediators.

Antivirals: lopinavir/ritonavir, arbidol, ribavirin, remdesivir,favipiravir, sofosbuvir and type I IFN. Ribavirin has been used inassociation with lopinavir/ritonavir to treat SARS-CoV-1 and otherantivirals such as sofosbuvir can strongly bind to coronavirus.Remdesivir (RDV) originally developed to treat Ebola virus infection,RDV is active against RNA viruses from different families, includingCoronaviridae (e.g., SARS-CoV-1 and MERS-CoV). IFN-α2b has been used inmany regimens including stand alone protocols.

Parenteral or enteral malaria chemoprevetion: chloroquine andhydroxychloroquine with or without macrolide antibiotics. Part of theWHO model list of essential medicines, immunomodulatory they also showbroad-spectrum antiviral effects. Hydroxychloroquine-azithromycincombinations have been used.

Parenteral, or enteral antihelminthics: ivermectin, niclosamide,nitazoxanide. Ivermectin, the best known and most widely usedantiparasitic drug in human and veterinary medicine has been triedagainst SARS-CoV-2. Niclosamide, an old anthelmintic used to treattapeworm infections, and an antiprotozoal agent, nitazoxanide is anotherbroad-spectrum antiviral agent considered for treatment of COVID-19.

Parenteral, or enteral anticoagulants: heparin, warfarin, clopidogrel,rivaroxaban, ticagrelor, fondapariniux, argatroban. A high mortalityrisk in severe COVID-19 patients related to coagulopathy. In addition,heparin has an anti-inflammatory effect that can bind to inflammatorycytokines, chemokines, and proinflammatory proteins, inhibitingneutrophil chemotaxis and leukocyte migration

Parenteral, or enteral broad spectrum corticosteroids: dexamethasone,methylprednisolone, prednisolone, prednisone and hydrocortisone. Theseare broad anti-T-cell therapies. There is good evidence for use ofdexamethasone early.

Other Drugs used as antivirals or as anti-inflammatory agents. Theseinclude colchicine, sotretinoin, apremilast, and zanubrutinib.

Emricasan (a pan-caspase) inhibitor is also contemplated as atherapeutic agent for COVID-19 (see, e.g.,https://www.medrxiv.org/content/10.1101/2020.11.02.20223636v1.full andhttps://www.globenewswire.com/news-release/2020/10/27/2115049/0/en/Histogen-and-Amerimmune-Enter-into-a-Collaborative-Development-and-Commercialization-Agreement-for-Emricasan-in-the-Treatment-of-COVID-19.html).

Other potential therapeutic agents for COVID-19 treatment are summarizedby Shagufta (European Journal of Medicinal Chemistry 213 (2021) 113157https://doi org/10.1016/j.ejmech.2021.113157), which includeantiogensin-converting enzyme-2 (ACE-2) inhibitors such as chloroquineand hydroxychloroquine, N-(2-aminoethyl)-1 aziridine-ethanamine,GW280264X, TAPI-0 and TAPI-2, MLN-4760, HTC and HMHTCC, telmisartan, andrecombinant human antiotensin-converting enzyme 2; SARS-CoV-2 3CLprotease inhibitors such as lopinavir and ritonavir, N3, Compound 21 and22, α-ketoamides, and N-substituted isatin compounds; spike glycoproteininhibitors such as S1 subunit: RBD inhibitors such as SSAA09E2, K22 andrilapladib, S2 inhibitors such as TGG, luteolin and quercetin, ADS-J1and arbidol; cathepsin L proteinase inhibitors such as CID 23631927 andCID 16725315, SSAA09E1, MDL28170, and K11777; transmembrane serineprotease 2 inhibitors such as camostat; furin cleavage site inhibitorssuch as decanoyl-RVKR-chloromethylketone; and RNA-dependent RNApolymerase inhibitors such as remdesir and favipiravir.

van den Berg and to Velde ((2020) Severe COVID-19: NLRP3 InflammasomeDysregulated. Front. Immunol. 11:1580. doi: 10.3389/fimmu.2020.01580)propose a two pronged treatment: to reduce virus entry and help toeradicate the virus by boosting the immune system and in aninflammation-driven damaging phase the endogeneous adjuvant reaction ofthe immune system should be suppressed. Potential targets include anNLRP3 inflammasome inhibitor, HMGB1, reducing the number of neutrophils,and blocking downstream mediators of NLRP3, such as caspase-1 andcytokines IL-1β and IL-18 and their receptors.

de Rivero Vaccari et al. ((2020) The Inflammasome in Times of COVID-19.Front. Immunol. 11:583373. doi: 10.3389/fimmu.2020.583373) providetherapies targeting the inflammasome that could be used in the care ofCOVID-19 patients that are presented below:

Therapy Mechanism of action Dexamethasone Decreases airway inflammationby inhibiting NLRP inflammasome activation and levels of IL-1β and IL-18Enoxaparin Low molecular weight heparin shown to inhibit inflammasomeactivation in a mouse model of brain injury-induced ALI. IFN- β Type IIFNs decreased NLRP3 inflammasome activation through STAT1 MCC950Inhibits inflammasome activation by preventing NLRP3 oligomerizationIC100 Inhibits inflammasome activation by preventing ASC-speck formationM5049 Inhibits TLR7 and TLR8, which have been described in inflammasomeactivation Anakinra IL-1 receptor blocker Tocilizumab Therapeuticmonoclonal antibody that blocks IL-6 signaling

Yap et al. (J Immunol 2020; 205:307-312; Prepublished online 3 Junehttp://www.jimmunol.org/content/205/2/307 doi: 10.4049/jimmunol.20005132020) discuss available pharmaceutical agents that target a criticalcomponent of inflammasome activation, signaling leading to cellularpyroptosis, and the downstream cytokines as a promising target for thetreatment of severe coronavirus disease 2019-associated diseases.

Lopez-Reyes et al. ((2020) Front. Immunol. 11:570251. doi:10.3389/fimmu.2020.570251) review the molecular mechanisms by whichobesity-associated systemic inflammation could cause a more severeclinical presentation of COVID-19 and hypothesize he SARS-CoV-2infection could potentiate or accelerate the pre-existing systemicinflammatory state of individuals with obesity, via the NLRP3inflammasome activation and the release of pro-inflammatory cytokinesfrom cells trough Gasdermin-pores commonly found in cell death bypyroptosis.

Quagliariello et al. (European Review for Medical and PharmacologicalSciences 2020; 24: 9169-9171) argue that targeting NLRP3 inflammasomecould be a strategy to prevent cardiovascular outcomes [fulminantmyocarditis, heart failure, venous thoromboembolism (VTE)] and acuterespiratory distress syndrome (ARDS) in patients with SARSCoV-2infection. They discuss the rational for NLRP3 targeting in clinicaltrials as an effective therapeutic strategy aimed to improve prognosisof COVID-19, analyzing the potential of two therapeutic options(tranilast and OLT1177) currently available in clinical practice.

Lopes et al. (medRxiv preprint doi:https://doi.org/10.1101/2020.08.06.20169573) evaluate the addition ofcolchicine to standard treatments for COVID-19.

Combined use of different treatments in COVID-19 are presented in Table2 of Peng et al., Aging and Disease Volume 12, Number 1, February 2021and reproduced below.

Treatment Drug Drug target Dosage Source Arbidol + Arbidol Membranefusion inhibitor PO NCT04273763 (CN) Bromhexine Bromhexine ExpectorantPO Arbidol + Arbidol Membrane fusion inhibitor PO, 200 mg, tid, 14 dNCT04254874 (CN) IFN-β1α IFN- β1α Immunomodulator INH, 14 dChloroquine + Chloroquine Membrane fusion inhibitor and PO NCT04382846(EG) Ivermectin immunomodulator Ivermectin Importin (IMP) α/β receptorPO Chloroquine + Chloroquine Membrane fusion inhibitor and PO, 500 mg, 4d, 30 d NCT04399746 (MX) Ivermectin + immunomodulator Vitamin DIvermectin IMP α/β receptor PO, 6 mg, qd, Day 1, 7 and 8 Vitamin DVitamins PO, 400 UI bid Chloroquine + Chloroquine Membrane fusioninhibitor and PO, 450 mg, bid NCT04428268 (MX) Losartan immunomodulatorLosartan Angiotensin II receptor (type PO, 25 mg, bid AT1) antagonisChloroquine + Chloroquine Membrane fusion inhibitor and PO NCT04447534(EG) Zinc immunomodulator Zinc PO Hydroxychloroquine +Hydroxychloroquine —/Receptor binding and PO, 400 mg, bid, 7 dNCT04321278 (IL) Azithromycin membrane fusion inhibitor NCT04322123 (BR)Azithromycin Tetrcycline PO, 500 mg, qd NCT04329832 (US)Hydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 800 mg, qd NCT04347031 (RU) Azithromycin +/− immunomodulatortocilizumab Azithromycin Tetrcycline PO, 250 mg, bid TocilizumabAnti-IL-6R antibody IV Hydroxychloroquine + Hydroxychloroquine Membranefusion inhibitor and PO, 400 mg, bid, 5 d NCT04441424 (IQ)Azithromycin + immunomodulator Convalescent plasma AzithromycinTetrcycline PO, 500 mg, qd, 5 d Convalescent plasma Immunomodulator IV,400 mL, 5 d Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO, 200 mg, tid, 5 d NCT04338698 (BR) Azithromycin +immunomodulator NCT04338698 (PK) Oseltamivir Azithromycin TetrcyclinePO, 500 mg, Day 1; 250 mg, Day 2-5 Oseltamivir Nucleoside analog PO, 75mg, bid, 5 d Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO, 200 mg, tid NCT04341870 (FR) Azithromycin +immunomodulator Sarilumab Azithromycin Tetrcycline PO, 500 mg, Day 1;250 mg, Day 2-5 Sarilumab Anti-1L-6R antibody IV, 400 mg, Day 1Hydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 600 mg, Day 1, 200 NCT04528927 (TN) Azithromycin + zincimmunomodulator mg, Day 2-9 Azithromycin Tetrcycline PO, 500 mg, Day 1;250 mg, Day 2-5 Zinc PO, 220 mg, 10 d Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 200 mg, tid, 14 dNCT04373044 (US) Baricitinib immunomodulator Baricitinib JAK inhibitorPO, 2 mg, qd, 14 d Hydroxychloroquine + Hydroxychloroquine Membranefusion inhibitor and PO, 200 mg, tid NCT04273763 (CN) Bromhexineimmunomodulator NCT04340349 (MX) Bromhexine Expectorant PO, 8 mg, tidHydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 400 mg, bid, 10 d NCT04355052 (IL) Camostat mesylate immunomodulatorCamostat mesylate TMPRSS2 inhibitor PO, 200 mg, qd, 10 dHydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 200 mg, tid, 7 d NCT04349410 (US) Clindamycin immunomodulatorClindamycin Lincomycin antibiotics IV, 4800 mg Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 200 mg, tid, 7 dNCT04349410 (US) Clindamycin + immunomodulator Primaquine ClindamycinLincomycin antibiotics IV, 4800 mg, 7 d Primaquine Membrane fusioninhibitor and PO, 200 mg, qd, 7 d immunomodulator Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 400 mg, bid, 14 dNCT04443725 (EG) Daclatasvir + immunomodulator Sofosbuvir DaclatasvirNucleoside analog PO, 90 mg, qd, 14 d Sofosbuvir Nucleoside analog PO,400 mg, qd, 14 d Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO, 200 mg, tid, 10 d NCT04349410 (US) Doxy cy dineimmunomodulator Doxycycline Tetrcycline IV, 100 mg, bid, 10 dHydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 200 mg, bid, 7 d NCT04359615 (IR) Favipiravir immunomodulatorNCT04376814 (IR) Favipiravir Nucleoside analog PO, 1600 mg, Day 1, 600mg, tid, 7 d Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO NCT04273763 (CN) IFN-α2β immunomodulator IFN-α2βImmunomodulator INH Hydroxychloroquine + Hydroxychloroquine Membranefusion inhibitor and PO, 200 mg, bid NCT04346147 (ES) Imatinibimmunomodulator Imatinib TMPRSS2 inhibitor PO, 400 mg, qdHydroxychloroquine + Hydroxychloroquine Membrane fusion inhibitor andPO, 200 mg, bid, 7 d NCT04344457 (US) Indomethacin + immunomodulatorAzithromycin Indomethacin Nonsteroidal anti-inflammatory PO, 50 mg, tid,14 d drug Zithromax Tetrcycline PO, 500 mg, qd, 3 d Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO NCT04350684 (IR)Lopinavir + immunomodulator NCT04343768 (IR) Ritonavir + LopinavirNucleoside analog PO NCT04350671 (IR) IFN-β1α Ritonavir Nucleosideanalog PO IFN-β1α Immunomodulator IV Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 200 mg, bidNCT04390152 (CO) Lopinavir + immunomodulator NCT04346147 (ES) RitonavirLopinavir Nucleoside analog PO, 200 mg, qd Ritonavir Nucleoside analogPO, 50 mg, qd Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO NCT04343768 (IR) Lopinavir + immunomodulatorRitonavir + Lopinavir Nucleoside analog PO IFN-β1β Ritonavir Nucleosideanalog PO IFN-β1β Immunomodulator PO Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 200 mg, bid, 5 dNCT04338698 (PK) Oseltamivir immunomodulator NCT04303299 (TH)Oseltamivir Nucleoside analog PO, 75 mg, bid, 5 d Hydroxychloroquine +Hydroxychloroquine Membrane fusion inhibitor and PO, 600 mg, 10 dNCT04374903 (JO) Sirolimus f immunomodulator Sirolimus ImmunosuppressantPO, 250 mg, 10 d Hydroxychloroquine + Hydroxychloroquine Membrane fusioninhibitor and PO, 200 mg, tid, 14 d NCT04390061 (US) Tofacitinibimmunomodulator Tofacitinib JAK inhibitor PO, 10 mg, bid, 14 dAzithromydn + Azithromycin Tetrcycline PO, 500 mg, Day 1; 250NCT04363060 (FR) Amoxicillin mg, Day 2-5, 2 d Amoxicillin/ClavulanateAntibacterial drugs PO, 250 mg, tid, 2 d Azithromycin + AzithromycinTetrcycline PO, 500 mg, Day 1; 250 NCT04339426 (US) Atovaquone mg, Day2-5 Atovaquone PO, 750 mg, bid, 10 d Azithromycin + AzithromycinTetrcycline PO, 250 mg, tid, 7 d NCT04363060 (FR) ClavulanateClavulanate Azithromycin + Azithromycin Tetrcycline PO, 500 mg, Day 1;250 NCT04528927 (TN) Doxycycline mg, Day 2-5 Doxycycline Tetrcycline PO,200 mg, qd, 10 d Azithromycin + Azithromycin Tetrcycline PO NCT04382846(EG) Ivermectin + Ivermectin IMP α/β receptor PO NitazoxanideNitazoxanide Immunomodulator PO Azithromycin + Azithromycin TetrcyclinePO, 250 mg, tid, 7 d NCT04347031 (RU) Mefloquine +/− Mefloquine Membranefusion inhibitor and PO, 500 mg, bid, 7 d Tocilizumab immunomodulatorTocilizumab Anti-IL-6R antibody IV Azithromycin + AzithromycinTetrcycline PO NCT04382846 (EG) Nitazoxanide NitazoxanideImmunomodulator PO Azithromycin + Azithromycin Tetrcycline PO, 500 mg,Day 1; 250 NCT04338698 (PK) Oseltamivir mg, Day 2-5 OseltamivirNucleoside analog PO, 75 mg, bid Azythromycin + Azythromycin TetrcyclinePO, 500 mg, qd NCT04446429 (BR) Ivermectin + Ivermectin IMP α/β receptorPO, 200 mcg/kg, qd Dutasteride Dutasteride TMPRSS2 inhibitor PO, 0.5 mgAzythromycin + Azythromycin Tetrcycline PO, 500 mg, qd NCT04446429 (BR)Ivermectin + Ivermectin IMP α/β receptor PO, 200 mcg/kg, qdProxalutamide Proxalutamide TMPRSS2 inhibitor PO, 200 mg Ivermectin +Ivermectin IMP α/β receptor PO, 200 mcg/kg, qd, 5 d NCT04407130 (BD)Doxycycline Doxycycline Tetrcycline PO, 200 mg, 5 d NCT04523831 (BD)NCT04403555 (EG) Ivermectin + Ivermectin IMP α/β receptor PO, 200mcg/kg, qd NCT04446429 (BR) Dutasteride + Dutasteride TMPRSS2 inhibitorPO, 0.5 mg Ivermectin + Ivermectin IMP α/β receptor PO, 12 mg, qd, 15 dNCT04447235 (BR) Losartan Losartan Angiotensin II receptor (type PO, 50mg, qd, 15 d AT1) antagonis Ivermectin + Ivermectin IMP α/β receptor PONCT04382846 (EG) Nitazoxanide Nitazoxanide Immunomodulator POIvermectin + Ivermectin IMP α/β receptor PO, 7 d NCT04392427 (EG)Nitazoxanide + Nitazoxanide Immunomodulator PO, 7 d Ribavirin RibavirinNucleoside analog PO, 7 d Ledipasvir + Ledipasvir Nucleoside analog PO,90 mg, qd, 14 d NCT04498936 (EG) Sofosbuvir Sofosbuvir Nucleoside analogPO, 400 mg, qd, 14 d Daclatasvir + Daclatasvir Nucleoside analog PO, 120mg, Day 1; 60 NCT04468087 (BR) Sofusbuvir mg, Day 2-9 NCT04460443 (EG)Sofusbuvir Nucleoside analog PO, 800 mg, Day 1; 400 mg, Day 2-9Danoprevir + Danoprevir Nucleoside analog PO, 100 mg, bid, 10 dNCT04345276 (CN) Ritonavir Ritonavir Nucleoside analog PO, 100 mg, bidNCT04291729 (CN) Darunavir + Darunavir Viral RNA-dependent RNA PO, 800mg, qd, 5 d NCT04252274 (CN) Cobicistat polymerase inhibitor/CYP3ANCT04425382 (QA) protein inhitor Cobicistat Protease inhibitor PO, 150mg, qd, 5 d Darunavir + Darunavir Nucleoside analog PO, 400 mg, tidNCT04303299 (TH) Ritonavir + Ritonavir Nucleoside analog PO, 200 mg, qdFavipiravir + Favipiravir Nucleoside analog PO, 2400 mg, qdHydroxychloroquine Hydroxychloroquine Membrane fusion inhibitor and PO,800 mg, qd immunomodulator Darunavir + Darunavir Nucleoside analog PO,400 mg, tid NCT04303299 (TH) Ritonavir + Ritonavir Nucleoside analog PO,200 mg, qd Oseltamivir + Oseltamivir Nucleoside analog PO, 300 mg, qdHydroxychloroquine Hydroxychloroquine Membrane fusion inhibitor and 8PO, 00 mg, qd immunomodulator Darunavir + Darunavir Nucleoside analogPO, 400 mg, tid NCT04303299 (TH) Ritonavir + Ritonavir Nucleoside analogPO, 200 mg, qd Oseltamivir Oseltamivir Nucleoside analog PO, 300 mg, qdEmtricitabine + Emtricitabine Protease inhibitor PO, 300 mg, qd, 8 dNCT04519125 (CO) Tenofovir Tenofovir Nucleoside analog PO, 200 mg, qdEmtricitabine + Emtricitabine Protease inhibitor PO, 200 mg, qdNCT04405271 (AR) Tenofovir Alafenamide Tenofovir alafenamide Nucleosideanalog PO, 25 mg, qd Favipiravir + Favipiravir Nucleoside analog PO, 200mg, qd, 10 d NCT04475991 (MX) Maraviroc Maraviroc Chemokine receptorantagonist PO, 300 mg, qd, 10 d Favipiravir + Favipiravir Nucleosideanalog PO, 1600 mg, Day 1,600 NCT04310228 (CN) Tocilizumab mg, tid, 6 dTocilizumab Anti-IL-6R antibody IV, 4-8 mg/kg, 7 d Oseltamivir +Oseltamivir RdRP inhibitor PO, 75 mg, qd, 14 d NCT04261270 (CN) ASC09FASC09F CYP3A4 inhibitor PO, 400 mg, bid, 14 d Oseltamivir + OseltamivirNucleoside analog PO, 4 w NCT04371601 (CN) Mesenchymal stem cellsMesenchymal stem cells MSC therapy IV, 1 × 10^({circumflex over ( )})6cell/kg/w, 4 w Oseltamivir + Oseltamivir Viral RNA-dependent RNA/ PO, 75mg, qd, 7 d NCT04315896 (MX) Ritonavir Booster of other proteaseNCT04318444 (US) polymerase inhibitor NCT04328285 (FR) RitonavirNucleoside analog PO, 300 mg, bid, 7 d Lopinavir + LopinavirAnti-retroviral of the protease PO, 400 mg, bid, 5 d Guidelines (version7) for Ritonavir inhibitor/booster of other treatment of COVID-19protease inhibitors Ritonavir Nucleoside analog PO, 100 mg, bid, 5 dNCT04328285 (FR) NCT04328012 (US) Lopinavir + Lopinavir Anti-retroviralof the protease PO, 400 mg, bid, 5-21 d NCT04350671 (IR) Ritonavir +inhibitor/booster of other NCT04403100 (BR) Arbidol protease inhibitorsNCT04376814 (IR) Ritonavir Nucleoside analog PO, 100 mg, bid, 5-21 dArbidol Membrane fusion inhibitor and PO, 200 mg, tid, 5-21 dimmunomodulator/Anti- retro viral of the protease inhibitor/booster ofother protease Lopinavir + Lopinavir Anti-retroviral of the protease PO,200 mg, qd, 10 d NCT04466241 (FR) Ritonavir + inhibitor/booster of otherAtorvastatin protease inhibitors Ritonavir Nucleoside analog PO, 50 mg,qd, 10 d Atorvastatin Statin medication PO, 20 mg, qd, 10 d Lopinavir +Lopinavir Anti-retroviral of the protease PO, 400 mg, bid, 7 dNCT04499677 (GB) Ritonavir + inhibitor/booster of other NCT04303299 (TH)Favipiravir protease inhibitors Ritonavir Nucleoside analog PO, 100 mg,bid, 7 d Favipiravir Nucleoside analog PO, 1800 mg, bid, Day 1; 400 mg,4 times, 7 d Lopinavir + Lopinavir Anti-retroviral of the protease PO,200 mg, qd NCT04315948 (FR) Ritonavir + inhibitor/booster of otherNCT04276688 (CN) IFN-β1α protease inhibitors Ritonavir Nucleoside analogPO, 50 mg, qd IFN-β1α Immunomodulator INH, 44 μg/0.5 mL Lopinavir +Lopinavir Anti-retroviral of the protease PO, 800 mg, qd NCT04303299(TH) Ritonavir + inhibitor/booster of other Oseltamivir proteaseinhibitors Ritonavir Nucleoside analog PO, 200 mg, qd OseltamivirNucleoside analog PO, 300 mg, qd Lopinavir + Lopinavir Anti-retroviralof the protease PO, 200 mg, qd, 10 d NCT04466241 (FR) Ritonavir +inhibitor/booster of other Telmisartan protease inhibitors RitonavirNucleoside analog PO, 50 mg, qd, 10 d Telmisartan Angiotensin IIreceptor (type PO, 40 mg, qd, 10 d ATI) antagonis Lopinavir + LopinavirAnti-retroviral of the protease PO, 400 mg, bid, 14 d NCT04276688 (CN)Ritonavir + inhibitor/booster of other NCT04343768 (IR) Ribavirin +protease inhibitors IFN-β1α Ritonavir Nucleoside analog PO, 100 mg, bid,14 d Ribavirin Nucleoside analog PO, 400 mg, bid, 14 d IFN-β1αImmunomodulator SC, 0.25 mg Remdesivir + Remdesivir Nucleoside analogPO, 200 mg, qd, Day 1; NCT04488081 (US) Apremilast 100 mg, qd, Day 2-9Apremilast Antiemetic PO, 30 mg, bid, 9 d Remdesivir + RemdesivirNucleoside analog PO, 200 mg, qd, Day 1; NCT04488081 (US) Cenicriviroc100 mg, qd, Day 2-9 Cenicriviroc CCR5 inhibitor PO, 150 mg, bid, 28 dRemdesivir + Remdesivir Nucleoside analog PO, 200 mg, qd, Day 1;NCT04401579 (US) Baricitinib 100 mg, qd, Day 2-9 Baricitinib JAKinhibitor PO, 4 mg, qd, 14 d Remdesivir + Remdesivir Nucleoside analogPO, 200 mg, qd, Day 1; NCT04488081 (US) Icatibant 100 mg, qd, Day 2-9Icatibant Peptide-based hormone SC, 30 mg, 9 d Remdesivir + RemdesivirNucleoside analog PO, 200 mg, qd, Day 1; NCT04492475 (US) IFN-β1α 100mg, qd, Day 2-9 IFN-β1α Immunomodulator SC, 44 μg/0.5 mL Remdesivir +Remdesivir Nucleoside analog PO, 200 mg, qd, Day 1; NCT04410354 (US)Merimepodib 100 mg, qd, Day 2-9 Merimepodib Inosine monophosphate IV,400 mg, tid, 10 d dehydrogenase (IMPDH) inhibitor Remdesivir +Remdesivir Nucleoside analog PO, 1 mg/kg NCT04480333 (US) NA-831 NA-831Endogenous small molecule INH, 0.2 mg/kg Remdesivir + RemdesivirNucleoside analog PO, 200 mg, qd, Day 1; NCT04488081 (US) Razuprotafib100 mg, qd, Day 2-9 Razuprotafib VE-PTP inhibitor SC, 10 mg, tid, 7 dRemdesivir + Remdesivir Nucleoside analog PO, 10 d NCT04409262 (US)Tocilizumab Tocilizumab Anti-IL-6R antibody IV, 10 d Ribavirin +Ribavirin Nucleoside analog PO NCT04460443 (EG) Sofosbuvir SofosbuvirNucleoside analog PO Ritonavir + Ritonavir Nucleoside analog PO, 100 mg,bid, 14 d NCT04261907 (CN) ASC09 ASC09 Protease inhibitors PO, 300 mg,bid, 14 d Ritonavir + Ritonavir Nucleoside analog PO, 100 mg, bid, 14 dNCT04291729 (CN) Ganovo + Danoprevir Nucleoside analog PO, 100 mg, bid,14 d IFN-Nebulization IFN-Nebulization Immunomodulator INH, 50 μg, bid,14 d IFN- α1β + IFN- α1β Immunomodulator ISIN, 2-3 drops, 4 timesNCT04320238 (CN) Thymosin α1 Thymosin α1 Immunomodulator SC, 1 time perweek IFN-β1β + IFN-β1β Immunomodulator SC or IV, 16 million UI,NCT04465695 (CN) clofazimine 3 d Clofazimine Nucleoside analog PO, 100mg, bid Adalimumab + Adalimumab Humanized monoclonal Adalimumab: SC, 40mg, ChiCTR2000030580 Tocilizumab antibody against the TNF- alpha every 2weeks; Tocilizumab: IV, 8 mg/kg, 6 times in 4 weeks TocilizumabAnti-IL-6R antibody IV C486 — SC or IV REGN10933 + REGN10933 Anti-Spike(S) SARS-CoV- 2 SC or IV NCT04426695 (US) REGN10987 REGN10987 antibodyNCT04519437 (US) NCT04452318 (US) Tocilizumab + Tocilizumab Anti-1L-6Rantibody IV, 8 mg/kg, Day 1 and 3 NCT04476979 (GF) DexamethasoneDexamethasone Corticosteroids IV, 10 mg for 5 d, 2.5 mg for 4 dTocilizumab + Tocilizumab Anti-IL-6R antibody IV, 8 mg/kg, Day 1 and 3NCT04377503 (ES) Methylprednisolone Methylprednisolone CorticosteroidsIV, 1.5 mg/kg/d, 21 d Tocilizumab + Tocilizumab Anti-IL-6R antibody IV,8 mg/kg NCT04335305 (ES) Pembrolizumab Pembrolizumab PD-1 antibody IV,200 mg Toremifene + Toremifene Hormone PO, 60 mg, qd NCT04531748 (US)Melatonin Melatonin Hormone PO, 40 mg, morning; 60 mg, eveningAnakinra + Anakinra 1L antagonists IV, 300 mg/d, 5 d NCT04366232 (FR)Ruxolitinib Ruxolitinib JAK inhibitor PO, 5 mg, bid, 14-28 d Anakinra +Anakinra 1L antagonists SC, 100 mg, qd NCT04330638 (BE) SiltuximabSiltuximab Anti-IL-6R antibody IV, 11 mg/kg Anakinra + Anakinra 1Lantagonists SC, 100 mg, qd NCT04330638 (BE) Tocilizumab TocilizumabAnti-IL-6R antibody IV, 8 mg/kg Colchicine + Colchicine NLRPInflammasome inhibitor PO, 0.5 mg, qd NCT04516941 (CH) Edoxaban EdoxahanThrombolytic medication PO, 60 mg, qd Colchicine + Colchicine NLRPInflammasome inhibitor PO, 0.5 mg, qd, 14 d NCT04492358 (ES)Methylprednisolone Methylprednisolone Corticosteroids PO, 60 mg, qd, 3 dColchicine + Colchicine NLRP Inflammasome inhibitor PO, 0.6 mg, qd, 3 dNCT04472611 (US) Rosuvastatin Rosuvastatin HMG-CoA reductase inhibitorsPO, 40 mg, qd, 3 d Dexamethasone + Dexamethasone Corticosteroids IVNCT04461925 (UA) Placenta-Derived MMSC Placenta-Derived MMSCs MSCtherapy IV, 1 × 10{circumflex over ( )}6 cell/kg, Day 1, 4 and 7Diltiazem + Diltiazem Calcium-channel blocker PO, 500 mg × 4 times, 10 dNCT04372082 (FR) Niclosamide Niclosamide — PO, 60 mg, tid, 10 dDipyridamole + Dipyridamole Thrombolytic medication PO, 200 mg, qd, 14 dNCT04410328 (US) Aspirin Aspirin Thrombolytic medication PO, 25 mg, qd,14 d Enoxaparin + Enoxaparin Thrombolytic medication SC, 4000-6000 UINCT04528888 (IT) Methylprednisolone Methylprednisolone CorticosteroidsIV, 0.5 mg/kg Heparin + Heparin Thrombolytic medication IV, 18 Ul/kg/hNCT04485429 (BR) Methylprednisolone Methylprednisolone CorticosteroidsIV, 0.5 mg/kg NCT04528888 (IT) Heparin + Umbilical Heparin Thrombolyticmedication IV NCT04355728 (US) Cord Mesenchymal Stem Umbilical Cord MSCtherapy IV, 100 × 10{circumflex over ( )}6 cell Cells Mesenchymal StemCells Levamisole + Levamisole — PO, 50 mg, tid, 14 d NCT04383717 (EG)Isoprinosine Isoprinosine — PO, 1 g, 4 times NCT04360122 (EG)Metenkefalin + Metenkefalin Opioid delta receptor agonists IV, 5 mgNCT04374032 (YU) Tridecactide Tridecactide Th1 cell modulators IV, 1 mgNitazoxanide + Nitazoxanide Immunomodulator PO, 1000 mg, bid NCT04459286(NG) atazanavir + Atazanavir Nucleoside analog PO, 300 mg, qd ritonavirRitonavir Nucleoside analog PO, 100 mg, qd Paracetamol + ParacetamolNon-steroidal anti-inflammatory PO, NCT04324606 (GB) ChAdOxl drugsnCoV-19 ChAdOxl nCoV-19 Vaccine IV, 5 × 10{circumflex over ( )}10 vpParacetamol + Paracetamol Non-steroidal anti-inflammatory IV NCT04324606(GB) MenACWY drugs MenACWY Vaccine IV Ruxolitinib + Ruxolitinib JAKinhibitor PO, 5 mg, bid, 14 d NCT04348695 (ES) Simvastatin SimvastatinStatin medication PO, 40 mg, qd, 14 d SnPP Protoporphyrin + SnPPProtoporphyrin Photodynamic therapy IV, 5, 7 and 9 mg, 14 d NCT04371822(EG) Sunlight exposure Sunlight exposure 1 h, 14 dSulfonatoporphyrin(TPPS) + Sulfonatoporphyrin(TPPS) Photodynamic therapyIV, 5 mg, 14 d NCT04371822 (EG) Sunlight exposure Sunlight exposure 1 h,14 d Tacrolimus + Tacrolimus Immunosuppressant PO, 8-10 ng/mL bloodNCT04341038 (ES) Methylprednisolone level MethylprednisoloneCorticosteroids PO, 120 mg, 3 d Tirofiban + Clopidogrel + TirofibanThrombolytic medication IV, 0.15 μg/kg/min NCT04368377 Acetylsalicylicacid + Clopidogrel Thrombolytic medication PO, initial dose of 300Fondaparinux mg, then 75 mg/d Acetylsalicylic add Thrombolyticmedication IV, initial dose of 75 mg, then 30 mg/d FondaparinuxThrombolytic medication IV, 2.5 mg PO: Oral administration; IV:Intravenous administration; INH: Inhalation; IM: intramuscularadministration; SC: Subcutaneous administration; ISIN: Intrasinaladministration; ID: intradermal injection; SL: Sublingualadministration; TOPp: patch applied on the skin; ET: Endotrachealinstillation

Pharmaceutical compositions of this invention may comprise a compound ofVX-765 or any other caspase 1/ICE inhibitor or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier. Suchcompositions may optionally comprise an additional therapeutic agent.Such agents include, but are not limited to, an anti-inflammatory agent,a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandin,or an anti-vascular hyperproliferation compound.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier that may be administered to a patient, together with a compoundof this invention, and which does not destroy the pharmacologicalactivity thereof.

Pharmaceutically acceptable carriers that may be used in thepharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat and self-emulsifying drug delivery systems (SEDDS) such as.alpha.-tocopherol, polyethyleneglycol 1000 succinate, or other similarpolymeric delivery matrices.

In pharmaceutical composition comprising only a caspase 1/ICE inhibitoras the active component, methods for administering these compositionsmay additionally comprise the step of administering to the subject anadditional agent. Such agents include, but are not limited to, ananti-inflammatory agent, a matrix metalloprotease inhibitor, alipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, ananti-cancer agent, an anti-viral agent, a cytokine, a growth factor, animmunomodulator, a prostaglandin, or an anti-vascular hyperproliferationcompound.

The term “pharmaceutically effective amount” refers to an amounteffective in treating or ameliorating an IL-1-, apoptosis-, other formsof cell death such as pyroptotic cell death and necrotic cell death-,IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated disease in a patient. Theterm “prophylactically effective amount” refers to an amount effectivein preventing or substantially lessening IL-1-, apoptosis-, other formsof cell death such as pyroptotic cell death and necrotic cell death-,IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated disease in a patient.

The compounds of this invention may be employed in a conventional mannerfor controlling IL-18 and IFN-γ levels in vivo and for treating diseasesor reducing the advancement or severity of effects which are mediated byIL-1, apoptosis, other forms of cell death such as pyroptotic cell deathand necrotic cell death, IL-6, IL-17, IL-18, IFN-γ or TNF-α-. Suchmethods of treatment, their dosage levels and requirements may beselected by those of ordinary skill in the art from available methodsand techniques.

For example, a compound of this invention may be combined with apharmaceutically acceptable adjuvant for administration to a patientsuffering from an IL-1-, apoptosis-, other forms of cell death such aspyroptotic cell death and necrotic cell death, IL-6, IL-17-, IL-18-,IFN-γ- or TNF-α-mediated disease in a pharmaceutically acceptable mannerand in an amount effective to lessen the severity of that disease.

Alternatively, the compounds of this invention may be used incompositions and methods for treating or protecting individuals againstIL-1, apoptosis-, other forms of cell death such as pyroptotic celldeath and necrotic cell death, IL-6, IL-17-, IL-18-, IFN-γ- orTNF-α-mediated diseases over extended periods of time. The compounds maybe employed in such compositions either alone or together with othercompounds of this invention in a manner consistent with the conventionalutilization of enzyme inhibitors in pharmaceutical compositions. Forexample, a compound of this invention may be combined withpharmaceutically acceptable adjuvants conventionally employed invaccines and administered in prophylactically effective amounts toprotect individuals over an extended period of time against IL-1-,apoptosis-, other forms of cell death such as pyroptotic cell death andnecrotic cell death, IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediateddisease.

The caspase 1/ICE inhibitor(s) may also be co-administered with othercaspase 1 inhibitors to increase the effect of therapy or prophylaxisagainst various IL-1-, apoptosis-, other forms of cell death such aspyroptotic cell death and necrotic cell death, IL-6, IL-17-, IL-18-,IFN-γ- or TNF-α-mediated diseases.

In addition, the compounds of this invention may be used in combinationwith either conventional anti-inflammatory agents or with matrixmetalloprotease inhibitors, lipoxygenase inhibitors and antagonists ofcytokines other than IL-1β.

The compounds of this invention can also be administered in combinationwith immunomodulators (e.g., bropirimine, anti-human alpha-interferonantibody, IL-2, GM-CSF, anti-GMCSF monoclonal antibodies (such as, forexample Gimsilumab from Roivant), methionine enkephalin,interferon-alpha, diethyldithiocarbamate, tumor necrosis factor,naltrexone, and EPO), with prostaglandins, or with antiviral agents(e.g., 3TC, remdesivir, polysulfated polysaccharides, ganiclovir,ribavirin, acyclovir, alpha interferon, trimethotrexate, andfancyclovir) or prodrugs of these or related compounds to prevent orcombat IL-1-mediated disease symptoms such as inflammation.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to the patient. Alternatively, pharmaceutical orprophylactic compositions according to this invention comprise acombination of a caspase 1/ICE inhibitor and another therapeutic orprophylactic agent.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, or via an implanted reservoir. In an advantageousembodiment, the administration is oral administration. Thepharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intralesional, and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as those described in Pharmacopeia Helvetica, or asimilar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions and solutions and propylene glycol are administeredorally, the active ingredient is combined with emulsifying andsuspending agents. If desired, certain sweetening and/or flavoringand/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax, and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-administered transdermalpatches are also included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between 0.5 and about 75 mg/kg body weight per day andmost preferably between about 1 and 50 mg/kg body weight per day of theactive ingredient compound are useful in a monotherapy for theprevention and treatment of IL-1-, apoptosis-, other forms of cell deathsuch as pyroptotic cell death and necrotic cell death, IL-6, IL-17-,IL-18-, IFN-γ- or TNF-α-mediated diseases, including uveitis,inflammatory diseases, autoimmune diseases, destructive bone disorders,proliferative disorders, infectious diseases, degenerative diseases,necrotic diseases, inflammatory peritonitis, osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, adult respiratory distresssyndrome, glomerulonephritis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia,chronic active hepatitis, myasthenia gravis, inflammatory bowel disease,Crohn's disease, psoriasis, atopic dermatitis, graft vs. host disease,osteoporosis, multiple myeloma-related bone disorder, leukemias andrelated disorders, myelodysplastic syndrome, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer'sdisease, Parkinson's disease, cerebral ischemia, myocardial ischemia,myocardial infarction, congestive heart failure, Huntington's disease,atherosclerosis, spinal muscular atrophy, multiple sclerosis,AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia,neurological damage due to stroke, ulcerative colitis, infectioushepatitis, juvenile diabetes, lichen planus, acute dermatomyositis,eczema, primary cirrhosis, uveitis, Behcet's disease, atopic skindisease, pure red cell aplasia, aplastic anemia, amyotrophic lateralsclerosis, nephrotic syndrome and systemic diseases or diseases witheffects localized in the liver or other organs having an inflammatory orapoptotic component caused by excess dietary alcohol intake or viruses,such as HBV, HCV, HGV, yellow fever virus, dengue fever virus, andJapanese encephalitis virus.

Typically, the pharmaceutical compositions of this invention isadministered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form varies dependingupon the host treated and the particular mode of administration. Atypical preparation contain from about 5% to about 95% active compound(w/w). Preferably, such preparations contain from about 20% to about 80%active compound.

When the compositions of this invention comprise a combination of acaspase 1/ICE inhibitor and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 10% to 80% of the dosagenormally administered in a monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient depends upon a variety of factors, includingthe activity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, drug combination, the severity and course of the disease, andthe patient's disposition to the disease and the judgment of thetreating physician.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated diseases which may betreated or prevented by the compounds of this invention include, but arenot limited to, inflammatory diseases, autoimmune diseases,proliferative disorders, infectious diseases, and degenerative diseases.The apoptosis-mediated diseases which may be treated or prevented by thecompounds of this invention include degenerative diseases.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell deathmediated inflammatory diseases whichmay be treated or prevented include, but are not limited toosteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, andadult respiratory distress syndrome. Preferably the inflammatory diseaseis osteoarthritis or acute pancreatitis.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated autoimmune diseases whichmay be treated or prevented include, but are not limited to,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis,insulin-dependent diabetes mellitus (Type I), autoimmune hemolyticanemia, autoimmune neutropenia, thrombocytopenia, chronic activehepatitis, myasthenia gravis, multiple sclerosis, inflammatory boweldisease, Crohn's disease, psoriasis, atopic dermatitis and graft vs.host disease. Preferably the autoimmune disease is rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, psoriasis, or atopicdermatitis.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated destructive bone disorderswhich may be treated or prevented include, but are not limited to,osteoporosis and multiple myeloma-related bone disorder.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated proliferative diseases whichmay be treated or prevented include, but are not limited to, leukemiasand related disorders, such as myelodysplastic syndrome, acutemyelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma,Kaposi's sarcoma, and multiple myeloma.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated infectious diseases whichmay be treated or prevented include, but are not limited to, sepsis,septic shock, and Shigellosis.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell deaths mediated degenerative or necroticdiseases which may be treated or prevented by the compounds of thisinvention include, but are not limited to, Alzheimer's disease,Parkinson's disease, cerebral ischemia, and myocardial ischemia.Preferably, the degenerative disease is Alzheimer's disease.

IL-1, IL-17 or apoptosis other forms of cell death such as pyroptoticcell death and necrotic cell death mediated degenerative diseases whichmay be treated or prevented by the compounds of this invention include,but are not limited to, Alzheimer's disease, Parkinson's disease,cerebral ischemia, myocardial ischemia, spinal muscular atrophy,multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis,aging, alopecia, and neurological damage due to stroke.

Other diseases having an inflammatory or apoptotic component may betreated or prevented by the compounds of this invention. Such diseasesmay be systemic diseases or diseases with effects localized in the liveror other organs and may be caused by, for example, excess dietaryalcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus,dengue fever virus, and Japanese encephalitis virus.

IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated disease which may betreated or prevented by the compounds of this invention include, but arenot limited to, inflammatory, infectious, autoimmune, proliferative,neurodegenerative and necrotic conditions.

IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated inflammatory diseasewhich may be treated or prevented include, but are not limited toosteoarthritis, acute pancreatitis, chronic pancreatitis, asthma,rheumatoid arthritis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, cerebral ischemia, myocardial ischemia and adultrespiratory distress syndrome. Preferably, the inflammatory disease isrheumatoid arthritis, ulcerative colitis, Crohn's disease, hepatitis oradult respiratory distress syndrome.

IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated infectious disease whichmay be treated or prevented include, but are not limited to infectioushepatitis, sepsis, septic shock and Shigellosis.

IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated autoimmune disease whichmay be treated or prevented include, but are not limited toglomerulonephritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves' disease, autoimmune gastritis, insulin-dependentdiabetes mellitus (Type I), juvenile diabetes, autoimmune hemolyticanemia, autoimmune neutropenia, thrombocytopenia, myasthenia gravis,multiple sclerosis, psoriasis, lichen planus, graft vs. host disease,acute dermatomyositis, eczema, primary cirrhosis, hepatitis, uveitis,Behcet's disease, atopic skin disease, pure red cell aplasia, aplasticanemia, amyotrophic lateral sclerosis and nephrotic syndrome.Preferably, the autoimmune disease is glomerulonephritis,insulin-dependent diabetes mellitus (Type I), juvenile diabetes,psoriasis, graft vs. host disease or hepatitis.

More preferred diseases or conditions which may be treated or preventedinclude rheumatoid arthritis, inflammatory bowel disease, includingCrohn's disease and ulcerative colitis, inflammatory peritonitis,amyotrophic lateral sclerosis, septic shock, pancreatitis, traumaticbrain injury, organ transplant rejection, osteoporosis, osteoarthritis,asthma, uveitis, psoriasis, Alzheimer's disease, myocardial infarction,congestive heart failure, Huntington's disease, atherosclerosis, atopicdermatitis, or leukemias and related disorders, such as myelodysplasticsyndrome or multiple myeloma.

Accordingly, one embodiment of this invention provides a method fortreating or preventing an IL-1 or apoptosis, pyroptosis or necrosismediated disease in a subject comprising the step of administering tothe subject any compound, pharmaceutical composition, or combinationdescribed herein and a pharmaceutically acceptable carrier.

Another embodiment of this invention provides a method for decreasingIL-18 production in a subject comprising the step of administering tothe subject any compound, pharmaceutical composition, or combinationdescribed herein and a pharmaceutically acceptable carrier.

Yet another embodiment of this invention provides a method fordecreasing IFN-γ production in a subject comprising the step ofadministering to the subject any compound, pharmaceutical composition,or combination described herein and a pharmaceutically acceptablecarrier.

Although this invention focuses on the use of the compounds disclosedherein for preventing and treating IL-1, apoptosis, pyroptosis ornecrosis-, IL-6, IL-17-, IL-18-, IFN-γ- or TNF-α-mediated disease, thecompounds of this invention can also be used as inhibitory agents forother cysteine proteases.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Adaptive Immune Deficiency in a Lethal Case ofCOVID-19 in a Liver Transplant Patient-Potential Role for Pyroptosis

The liver disease and transplant communities provide a window intopopulations that are especially important to study in light ofcoronavirus disease 2019 (COVID-19) as these patients typically havemany co-morbidities, including diabetes, obesity and related cardiacissues, cancer, and most notably end-stage organ failure. What theseco-morbidities have in common is underlying chronic inflammationinvolving activation of the inflammasome (Guo H, Callaway J B, Ting J P.Inflammasomes: mechanism of action, role in disease, and therapeutics.Nat Med 2015; 21:677-687). What these patients have in common is lethalexperiences with COVID-19 (Onder G, Rezza G, Brusaferro S. Case-FatalityRate and Characteristics of Patients Dying in Relation to COVID-19 inItaly. JAMA 2020). Putting these two factors together caused Applicantsto speculate that the inflammasome, and more specifically pyroptosis,are what may drive tragic outcomes for patients with co-morbidities inthe face of COVID-19. Applicants successfully explored this hypothesisin a recent liver transplant patient with several co-morbidities, whorapidly succumbed to the disease, and whose clinical course andimmunological phenotype are described in this observation.

A 65-year old woman, with diabetes, hypertension, obesity, cirrhosisfrom primary sclerosing cholangitis, and a MELD score of 40 received aliver transplant in early 2020 (FIG. 2A) and was put on standardimmunosuppression. 26 days post-transplant she was discharged into asub-acute rehab facility, where Applicants suspect she contractedSARSCoV-2 as it later turned out to home three COVID-19 cases. Two daysafter returning home from the facility, she presented emergently withfever, dry cough, and shortness of breath for two days and was testedfor COVID-19. A chest x-ray revealed multilobular interstitialinfiltrates (FIG. 2B). She rapidly desaturated, got intubated, andunderwent cardiopulmonary resuscitation for pulselessness, with returnof spontaneous circulation. She was started on ARDSnet ventilation andhypothermia protocols in intensive care. Laboratory results showedmarked lymphopenia and elevated inflammatory markers (FIG. 2A). She wastreated with Hydroxychloroquine and Azithromycin after confirmingCOVID-19 the next day. Her neurological exam and head CT upon rewarmingshowed evidence of anoxic brain injury. ARDS and poor neurologicalstatus persisted (FIG. 2B), and she succumbed to COVID-19 six days afteradmission.

Although evidence for activation of antigen presenting cells (APCs) andinvolvement of Type-1-interferon response, as indicated by upregulationof CD11b and CD38 on monocytes and tetherin on B-cells, suggested innateimmune activation there was a profound adaptive immune dysfunction (FIG.2A and FIG. 3 ). Notably, Applicants found no B-cell memory cells andabsence of SARS-CoV-2-specific IgG and IgM. Moreover, she exhibitedprofound T-cell lymphopenia, further underscored by absence ofT-regulatory cells, low T-follicular helper cells, overexpression ofCD38 in CD8 T-cells, and poor STAT-4 phosphorylation of CD4 T-cells uponIL-12 stimulation. Finally, high levels of lactate dehydrogenase (LDH)(FIG. 2A) and a global increase in caspase-1 staining (FIG. 3 ) inlymphocytes hinted at pyroptosis (Rayamajhi et al. Methods Mol Biol2013; 1040:85-90 and Doitsh et al. Nature 2014; 505:509-514), which mayexplain her poor immune response.

Applicants' findings may shed new light on why and how SARS-CoV-2infections cause such rapid lethality. T cell lymphopenia and lack ofCOVID specific IgM indicates that the adaptive immune system neverproperly kicked in. Caspase-1 overexpression in lymphocytes suggestspyroptosis as one of the possible mechanisms of T-cell depletion andfunctional abnormalities of other lymphocytes (Doitsh et al. Nature2014; 505:509-514). This finding, even though limited to one patientpresented here and potentially exacerbated by immunosuppressivemedication, brings Applicants' attention to a COVID-19 induced acuteimmunodeficiency, resulting from the necrotic cell death of lymphocytes.Applicants propose that the inflammatory response is secondary to thedanger signals from necrotic cell death of immune system cells,resulting in a heightened inflammation compared to the one induced bydying tissue cells (Matzinger P. Tolerance, danger, and the extendedfamily. Annu Rev Immunol 1994; 12:991-1045). The end result is likely aself-damaging shut down of the immune system that further fuels theinflammation created by the viral infection. Applicants provide databacked connection between caspase-1 and COVID-19, a connection withsignificant therapeutic implications as rather than inhibition of theinflammatory response, preventing the pyroptotic lymphocyte deathfueling the inflammation becomes more critical.

This patient was a 65-year-old woman with decompensated liver cirrhosisfrom primary sclerosing cholangitis complicated by jaundice, ascites,hepatic encephalopathy, and portal hypertension with esophageal varices,and a history of chronic portal vein thrombosis requiringanticoagulation with warfarin and recanalization with transjugularintrahepatic portosystemic shunt (TIPS). Her model for end-stage liverdisease (MELDNa) score at time of transplant was 40. Past medicalhistory included insulin dependent diabetes mellitus, obesity (body massindex (BMI) 32), hypertension, and gout. Past surgical history includedopen cholecystectomy, umbilical hernia repair, and hysterectomy.

She underwent orthotopic liver transplantation in early 2020 (seeSupplementary Table 1 for her pre-transplant laboratory panel, noted asday −38 from the onset of COVID-19 symptoms). She received an organ froma 19-year-old male brain-dead deceased donor. Donor/recipientcytomegalovirus (CMV) and Epstein-Barr virus (EBV) serologies were +/+and +/+, respectively. The liver transplant was performed in a standardfashion (vena cava replacement without veno-venous bypass) with standardportal vein and arterial anastomoses. Intraoperative findings includedsignificant portal hypertension with venous collaterals, dense adhesionsfrom prior open cholecystectomy, intraoperative right atrial thrombuswhich resolved with tissue plasminogen activator (tPA) lysis, andsignificant blood loss due to coagulopathy.

After successful liver transplantation the patient was transferred tothe intensive care unit (ICU) with external biliary drainage and openabdomen. After resolution of the coagulopathy, she returned to theoperating room (OR) 72 hours later in stable conditions for biliaryreconstruction and closure of the abdomen.

Induction immunosuppression consisted of methylprednisolone (1 gintravenous therapy (i.v.) post-operative day (POD) 0) and basiliximab(20 mg i.v. POD 2 and 5, respectively). Maintenance immunosuppressionregimen included tacrolimus (2 mg orally (p.o.) twice per day),mycophenolate-mofetil (1 g p.o. twice per day), and standard steroidtaper. The patient received standard anti-infectious prophylaxis withsulfamethoxazole-trimethoprim, nystatin, and ganciclovir.

Her initial post-transplant course was characterized by slownormalization of serum bilirubin levels and acute renal failurerequiring renal replacement therapy (both of which resolved prior todischarge; see also Supplementary Table 1 for her laboratory panel upondischarge, noted as day −12 from the onset of COVID-19 symptoms), andanemia requiring transfusion of two units of packed red blood cells(PRBCs). In addition, her post-transplant course was notable for mentalstatus changes responding to Quetiapine without intracranial abnormalimaging findings, temporary dysphonia from vocal cord paralysis,dysphagia requiring enteral tube feeding via naso-jejunal tube andClostridium difficile-negative diarrhea, which improved after resumptionof regular diet. Incidentally, the patient was found to have a chronicleft humeral neck fracture on routine post-operative chest x-ray, whichwas treated conservatively.

On POD 26, the patient was discharged to a subacute rehab (SAR) facilityin stable condition with normal allograft function (see SupplementaryTable 1 for her labs upon discharge, noted as day −12 from the onset ofCOVID-19 symptoms). She returned for routine follow ups over the comingweeks, which all showed normal labs, including (coincidently) on thevery day that she later reported first feeling symptoms later that night(see Supplementary Table 1 for her labs that day, noted as day 0 fromthe onset of COVID-19 symptoms).

Two days after discharge from the rehabilitation facility, she presentedto Applicants' emergency department with fever 37.6?, dry cough, andshortness of breath for two days. On presentation the peripheral oxygensaturation (S-02) was 96% on room air; her blood pressure and heart ratewhere within normal limits. A nasopharyngeal swab specimen was obtainedand sent for COVID-19 real-time PCR testing (see methods below for testrelevant details). A chest x-ray revealed multilobular bilateralinterstitial infiltrates (see FIG. 2B). While completing evaluation inthe emergency department, the patient rapidly desaturated (S—O₂ 20-30%)and became unresponsive requiring bedside intubation. She was found tobe pulseless and underwent immediate cardiopulmonary resuscitation,including chest compressions with return of spontaneous circulation.Upon transfer to intensive care ARDSnet ventilation and hypothermiaprotocols were initiated. Laboratory results showed marked lymphopeniaand elevated inflammatory markers (see FIG. 2A). Empiric antibiotics(Cefepime (2 g i.v. q12 h) and Vancomycin (1 g i.v.) were administeredfor potential community acquired pneumonia. Hydroxychloroquine (400 mgtwice per day followed by 200 mg twice per day) and Azithromycin (250 mgi.v. q24 h) were added the following day upon positive result of thenasopharyngeal swab test for COVID-19. The neurological exam and head CTfindings upon rewarming showed evidence of diffuse anoxic brain injury.Poor neurological status and ARDS requiring ventilation (Fi02>=60%)persisted over subsequent days. Repeat CT imaging of the head and chest(see FIG. 2B) showed no improvement in terms of anoxic brain injury andARDS, and she succumbed to COVID-19 six days after admission, eight daysafter first experiencing symptoms.

The patient was enrolled in a MedStar Georgetown Transplant Institutebiorepository study (IRB #2017-0365). Written informed consent wasobtained prior to inclusion into that study. Ethical utilization oftransplant organs and tissue abided by principles in the Declaration ofHelsinki. The donor organ was not obtained from an executed prisoner orotherwise institutionalized person.

Clinical specimens of the nasopharynx for SARS-CoV-2 testing wereobtained, handled, and processed via real-time polymerase chain reactionassay as previously reported by Applicants' hospital group (Iqbal et al.N Engl J Med 2020 and Corman et al. Euro dSurveill 2020; 25). Briefly,viral nucleic acid was extracted by the MagNA Pure 96 IVD automatedinstrument (ROCHE Diagnostics) from nasopharyngeal swab specimen.Real-Time PCR was performed using the COBAS® Z480 Real-Time PCR System(ROCHE Diagnostics).

Patient samples for clinical immunophenotyping were obtained on day 4and day 7 after onset of symptoms. Images shown in FIGS. 3A-E are fromday 7 sample. A healthy sample was used as a matched control. Sampleswere processed in accordance with Amerimmune's clinical safety SOPs.Appropriate PPE were used when processing samples.

Peripheral blood from venipuncture was drawn into EDTA and heparincoated vacutainer tubes (BD Bioscience) for clinical immunophenotyping.Whole blood collected in EDTA tubes was immunostained per the clinicalstandard immunophenotyping protocol (Amerimmune LLC, Fairfax, Va.). Thesamples were stained with the antibody combinations as indicated in thesupplemental Table 1 for 30 minutes at 4° C. Red blood cells were lysedusing BD FACS lysis solution (BD Bioscience, Jan Jose, Calif.) as permanufacture directions.

Peripheral blood mononuclear cells (PBMC) were separated from 2 mL ofwhole blood diluted 1:1 with phosphate buffered saline pH 7.2 (PBS)(Thermo Fisher Scientific, Carlsbad, Calif.) using Lymphoprep (Stem cellTechnologies, Cambridge, Mass.) and Accuspin tubes (Sigma-Aldrich, St.Louis, Mo.) as per manufactures directions. PBMCs' were washed in PBSand resuspended in 0.4 mL PBS. 100 μL of the PBMCs were immunostainedwith a mixture of antibodies as indicated in supplemental Table 2 at 4?for 1 hour. Cells were washed and resuspended in PBS prior toacquisition.

The antibodies utilized from Thermo Fisher Scientific were CD56 SB436[TULY56], CD45 eF506 [HI130], CD3 FITC [SK7], CD16 PE [B73.1], CD8PerCP-eF710 [SK1], CD14 PE-CY7 [61D3], CD4 APC [SK-3], CD20 APC-eF780[2H7] CD25 EF450 [CD25-4E3], CD57 FITC [TBol], TCRγ-δ PE [B1.1], CD4PerCP-eF710 [SK-3], CD3 PE-CY7 [SK7], TCR αβ APC [IP26], HLA-DR AF700[LN3], CD8 APC-eF780 [SL1], IgD SB436 [IA6-2], IgA FC Secondary AntibodyFITC, IgG FC Secondary Antibody PE, IgM PerCPeF710 [SA-DA4], CD19 PE-CY7[SJ25C1], CD27 APC [0323], CD5 FITC [UCHT2], CD21 PE [HB5], CD27PerCP-eF710 [O323], CD45RA FITC [HI100], CD45RO PerCPeF710 [UCHL1],CD294 APC [BM16], CD4 AF700 [RPA-T4], CD3 FITC [SK7], CD14 FITC [61D3],CD16 FITC [3G8], CD19 FITC [SJ25-C1], CD20 FITC [2H7], CD56 FITC[TULY56], CD34 FITC [4H11], CD11c PE [3.9], HLA-DR PerCP-EF710 [L243],CD303a APC [201A], CD4 SB600 [SK-3], CD45RA FITC [HI100], CD3 PE-CY7[SK7], CD8 AF700 [SK1], CCR5 APC [NP-6G4], CD25 APC [BC96], CD317 PE[26F8], IL-6 PECY7 [MQ2-13A5], MIP1-β APC [FL34Z3L].

The antibodies utilized from BD Bioscience were HLADR BV480 [G46-6],CD38 PerCPCY5.5 [HIT2], CD28 APC [CD28.2], CD45 APC H7 [2D1], CD278BV421 [DX29], CXCR5 PerCP-CY5.5 [RF8B2], CD127 BV480 [HIL-7R-M21],CD45RO PerCP-CY5.5 [UCHL1], CD20 APC-H7 [2H7], CD11b BV421 [ICRF44],CD16 FITC [NKP15], MIP1-α PE [11A3], HLA-DR PerPC-Cy5.5 [L243], PerforinAlexa488 [δG9], and Granzyme B PE [GB11]. TNF-α BV421 [Mab11] was fromBiolegend (San Diego, Calif.).

Apoptosis and pyroptosis were measured by flow cytometry usingfluorescent-labeled inhibitors of caspase probe assay, FLICA, as permanufactures protocol (Immunochemistry Technologies, Minneapolis,Minn.). FAM-FLICA probes specific for Caspase 1 and Caspase 3/7 weredirectly added to 100 μl PBMC, incubated for 1 hour at 37° C. Cells werewashed 3 times with wash buffer to remove unbound FLICA probes. Cellswere stained for CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4],CD45RO PerCP-EF710 [UCHL1] and Viability Dye 780 (Thermo FisherScientific, Carlsbad, Calif.) to identify viable CD45+CD3+CD4+CD45RO-Tcells. An additional singlet gate was incorporated in the gating schemeto identify singlets.

Whole blood was stimulated with IL-12 (100 ng/ml) for 0, 15, 30, 60 and120 minutes. Cells were stained as per the BD Phosflow protocol. Inbrief, cells were stained with CD3 APC [SK7], CD4 PE-CY7 [SK3], CD45ROBV421 [UCHL1] and p-STAT4 [38/p-Stat4]. CD3+CD4+ T cells were identifiedfrom the lymphocyte and singlet gate described previously. TheCD45RO/pSSTAT4 plots are gated on CD3+CD4+ T cells.

The samples were acquired on a 3 laser BD FACS Canto 10. CS&T beads (BDBioscience, San Jose, Calif.) were acquired daily to ensure consistentperformance of the Canto10. The BD FACS Canto 10 was cleaned with 10minutes of 10% bleach and water following acquisition of samples. TheCANTO10 utilized for this study has been validated for T, B, NK andDendritic cell immunophenotyping clinical diagnostic testing. Denovo FCSExpress v6 clinical edition (De Novo Software, Pasadena, Calif.) wasused for flow cytometric analyses.

Monocytes were identified by a standard gating strategy utilizing CD14,CD16 and HLADR to identify classical, intermediate and nonclassicalmonocytes. CD38 and CD11b MFI and percent positive were examined formCD14++CD16+ intermediate monocytes.

Lymphocytes were identified using a standard gating schematicincorporated gating of lymphocytes on a FSC/SSC plot and singlets on aFSC-A/FSC-H plot. From the lymphocyte plots, T cells were identified asCD45+ on a CD45/SSC plot. CD45+CD3+ cells were gated on a CD3/SSC plot.CD4+ and CD8+ T cells were identified by CD4/CD8 CD3+ gating. SubsequentT cell subpopulations as indicated in FIG. 3B were identified fromCD45+CD3+CD4+ or CD8+ cells.

Perforin and Granzyme B immune-phenotyping was done on CD8+ T cells andNK cells. Lymphocytes were identified as described previously on aFSC/SSC and FSCA/FSC-H plots. NK and T cells were identified by aCD56/TCR α/β plot. NK cells were identified as CD56+TCR α/β- and T cellswere identified as TCRα/β+CD56- and CD8+ on a CD8/SSC plot. Identifiedspecific populations were examined for perforin and granzyme staining,using an FMO control for both perforin and granzyme.

From the lymphocyte gating plots, B cells were identified as CD45+, thenCD20+ from a CD20/SSC plot. All B cell subpopulations were identifiedfrom the CD45+CD20+ population.

For SARS-CoV-2 IgG and IgM antibody detection, Applicants used fourlateral flow assay kits for COVID IgG and/or IgM (Dynamiker Technology,Tianjin, China; Wondfo, Guangdong, China; and Shenyang UnionBiotechnologies, Liaoning, China), one chemiluminescent kit for COVID-19IgG and IgM (Xincheng Biotechnologies, Sichuan, China), and ELISA forCOVID-19 IgG/IgM (Antibody Biopharma, Gaithersburg, Md., USA). Thosetests have a sensitivity in the range of 86% to 92% for known SARS-CoV-2positive patients based on manufacturer information and previousclinical data. None of them were FDA approved assay at the time of thetesting.

Supplementary Table S1 - Patient Clinical Laboratory Results Time afteronset (d) −38 −12 0 1 2 3 Event Liver Dis- First Positive trans- chargedsymp- Presents COVID- Variable Reference range plant to SAR toms at ED19 test Troponin (ng/ml)    0-0.045 0.171 0.024 Sodium (mmol/liter)137-145 135 142 136 141 139 Potassium (mmol/liter) 3.5-5.1 3.8 5.1 5.33.9 3.8 Chloride (mmol/liter)  98-107 109 115 104 113 110 Carbon dioxide(mmol/liter) 21-32 19 18 25 21 18 Blood urea nitrogen (mg/dl)  7-17 2649 23 14 25 Creatinine (mg/dl) 0.52-1.04 0.89 0.93 0.92 0.71 1.30Glucose (mg/dl)  65-140 482 129 185 160 123 Calcium (mg/dl)  8.5-10.17.7 8.5 8.7 7.6 7.7 Total protein (gm/dl) 6.3-8.2 5.2 5.5 6.7 5.4 5.6Globulin (gm/dl) 1.3-4.7 3.5 3.5 3.9 3.1 3.2 Aspartate aminotransferase(u/liter)  3-34 126 15 14 19 74 Alanine aminotransferase (u/liter) 15-41128 17 23 27 89 Anion gap (mmol/liter)  5-15 7 9 7 7 11 Albumin (gm/dl)3.5-5.0 1.7 2.0 2.8 2.3 2.4 Total bilirubin (mg/dl) 0.2-1.3 26.7 1.1 0.80.8 0.7 Alkaline phosphatase (u/liter)  45-117 530 126 87 72 76 Lactatedehydrogenase (u/liter)  84-246 411 Lactic acid (mmol/liter) 0.7-2.0 0.7<0.7 <0.7 Hemoglobin (gm/dl)  11-14.5 8.2 8.1 7.8 7.0 8.0 Hematocrit (%)34.5-44  24.9 26.4 25.6 22.7 25.0 Platelet count (k/μL) 145-400 45 135145 139 94 Time after onset (d) 4 5 6 7 8 Event Variable Reference rangeDeath Troponin (ng/ml)    0-0.045 Sodium (mmol/liter) 137-145 138 138141 142 147 Potassium (mmol/liter) 3.5-5.1 3.7 4.0 3.7 3.4 3.4 Chloride(mmol/liter)  98-107 108 109 112 113 118 Carbon dioxide (mmol/liter)21-32 15 16 17 16 14 Blood urea nitrogen (mg/dl)  7-17 28 33 39 43 49Creatinine (mg/dl) 0.52-1.04 1.43 1.68 1.66 1.58 1.59 Glucose (mg/dl) 65-140 146 178 169 184 249 Calcium (mg/dl)  8.5-10.1 7.9 8.0 8.2 8.28.7 Total protein (gm/dl) 6.3-8.2 6.0 5.8 6.0 6.4 6.4 Globulin (gm/dl)1.3-4.7 3.8 3.9 4.2 4.4 4.5 Aspartate aminotransferase (u/liter)  3-3471 52 55 57 51 Alanine aminotransferase (u/liter) 15-41 88 62 43 36 24Anion gap (mmol/liter)  5-15 15 13 12 13 15 Albumin (gm/dl) 3.5-5.0 2.21.9 1.8 2.0 1.9 Total bilirubin (mg/dl) 0.2-1.3 0.7 0.6 0.5 0.5 0.6Alkaline phosphatase (u/liter)  45-117 79 87 87 94 102 Lactatedehydrogenase (u/liter)  84-246 373 Lactic acid (mmol/liter) 0.7-2.0<0.7 <0.7 <0.7 <0.7 1.0 Hemoglobin (gm/dl)  11-14.5 8.2 7.8 7.3 8.7 8.4Hematocrit (%) 34.5-44  25.0 24.4 22.8 26.8 26.5 Platelet count (k/μL)145-400 81 107 93 104 113 Items highlighted in red are outside thereference range.

Supplementary Table S2 - Patient Immunological Data Results Time afteronset (d) −38 −12 0 1 2 3 Event Liver Dis- First Positive trans- chargedsymp- Presents COVID- Variable Reference range plant to SAR toms at ED19 test Troponin (ng/ml)    0-0.045 0.171 0.024 Sodium (mmol/liter)137-145 135 142 136 141 139 Potassium (mmol/liter) 3.5-5.1 3.8 5.1 5.33.9 3.8 Chloride (mmol/liter)  98-107 109 115 104 113 110 Carbon dioxide(mmol/liter) 21-32 19 18 25 21 18 Blood urea nitrogen (mg/dl)  7-17 2649 23 14 25 Creatinine (mg/dl) 0.52-1.04 0.89 0.93 0.92 0.71 1.30Glucose (mg/dl)  65-140 482 129 185 160 123 Calcium (mg/dl)  8.5-10.17.7 8.5 8.7 7.6 7.7 Total protein (gm/dl) 6.3-8.2 5.2 5.5 6.7 5.4 5.6Globulin (gm/dl) 1.3-4.7 3.5 3.5 3.9 3.1 3.2 Aspartate aminotransferase(u/liter)  3-34 126 15 14 19 74 Alanine aminotransferase (u/liter) 15-41128 17 23 27 89 Anion gap (mmol/liter)  5-15 7 9 7 7 11 Albumin (gm/dl)3.5-5.0 1.7 2.0 2.8 2.3 2.4 Total bilirubin (mg/dl) 0.2-1.3 26.7 1.1 0.80.8 0.7 Alkaline phosphatase (u/liter)  45-117 530 126 87 72 76 Lactatedehydrogenase (u/liter)  84-246 411 Lactic acid (mmol/liter) 0.7-2.0 0.7<0.7 <0.7 Hemoglobin (gm/dl)  11-14.5 8.2 8.1 7.8 7.0 8.0 Hematocrit (%)34.5-44  24.9 26.4 25.6 22.7 25.0 Platelet count (k/μL) 145-400 45 135145 139 94 Time after onset (d) 4 5 6 7 8 Event Variable Reference rangeDeath Troponin (ng/ml)    0-0.045 Sodium (mmol/liter) 137-145 138 138141 142 147 Potassium (mmol/liter) 3.5-5.1 3.7 4.0 3.7 3.4 3.4 Chloride(mmol/liter)  98-107 108 109 112 113 118 Carbon dioxide (mmol/liter)21-32 15 16 17 16 14 Blood urea nitrogen (mg/dl)  7-17 28 33 39 43 49Creatinine (mg/dl) 0.52-1.04 1.43 1.68 1.66 1.58 1.59 Glucose (mg/dl) 65-140 146 178 169 184 249 Calcium (mg/dl)  8.5-10.1 7.9 8.0 8.2 8.28.7 Total protein (gm/dl) 6.3-8.2 6.0 5.8 6.0 6.4 6.4 Globulin (gm/dl)1.3-4.7 3.8 3.9 4.2 4.4 4.5 Aspartate aminotransferase (u/liter)  3-3471 52 55 57 51 Alanine aminotransferase (u/liter) 15-41 88 62 43 36 24Anion gap (mmol/liter)  5-15 15 13 12 13 15 Albumin (gm/dl) 3.5-5.0 2.21.9 1.8 2.0 1.9 Total bilirubin (mg/dl) 0.2-1.3 0.7 0.6 0.5 0.5 0.6Alkaline phosphatase (u/liter)  45-117 79 87 87 94 102 Lactatedehydrogenase (u/liter)  84-246 373 Lactic acid (mmol/liter) 0.7-2.0<0.7 <0.7 <0.7 <0.7 1.0 Hemoglobin (gm/dl)  11-14.5 8.2 7.8 7.3 8.7 8.4Hematocrit (%) 34.5-44  25.0 24.4 22.8 26.8 26.5 Platelet count (k/μL)145-400 81 107 93 104 113 Items highlighted in red are outside thereference range.

Example 2: Potential Role of the Inflammasome in Transplant Patientswith COVID-19

Applicants respond to the Correspondence “COVID-19 and KidneyTransplantation” which shared observations from kidney transplantpatients at New York's Montefiore Medical Center and highlighted threethemes: comorbidities, inflammation, and low T-cell/lymphocyte counts.

Based on observations from patients at the MedStar Georgetown TransplantInstitute Applicants can both confirm and expand these observationsbased on 11 patients recently hospitalized due to COVID-19, 6 of whomwere kidney and liver transplant patients (see below table).

Table for 11 Patients Sorted By Disease Severity—Page 1 of 2

Patient 1 2 3 4 5 6 Transplant Info Organ type Liver & Kidney NA KidneyNA Kidney NA Immunosuppression at presentation FK, MMF NA FK, MMF NA FK,MMF NA Potential COVID-19 Risk Categories Age > 50 No Yes No Yes Yes YesGender F M M M M F Race/ethnicity AA Other AA AA AA Caucasian BMI  32 31  37  21  30  29 Comorbidities  3  4  2  3  4  5 Diabetes ✓ ✓ Obesity✓ ✓ ✓ ✓ Hypertension ✓ ✓ ✓ Heart disease ✓ ✓ Other PE Liver Nephro- DVTLiver disease, cirrhosis, lithiasis, COPD, ETOH Hyper- arthritis,lipidemia hyperlipidemia COVID-19 Course and Outcome Key symptoms Fever✓ ✓ ✓ ✓ at time of Cough ✓ ✓ ✓ presentation Dyspnea ✓ ✓ ✓ ✓Myalgia/lethargy ✓ ✓ ✓ ✓ Diarrhea ✓ ✓ ✓ Radiographic viral PNA detection✓ ✓ ✓ ✓ Treatment Hydroxychloroquine ✓ ✓ ✓ ✓ ✓ Azithromycin ✓ ✓ OutcomeHospitalization ✓ ✓ ✓ ✓ ✓ ✓ Supplemental O₂ ✓ ✓ ✓ ✓ Intubation/ICU ✓ Nodischarge by d 14 Death Inflammatory Markers (initial recorded value |highest recorded value) LDH (u/liter) [84-246] 246 | 286 337 | 368 203 |246 518 | 559 240 | 331 957 | 957 CRP (mg/L) [0.0-3.0] 43.0 | 43.0 11.5| 31.9 32.7 | 70.6 240 | 240  46.7 | 138.0 53.3 | 53.3 Ferritin (ng/mL)[5.0-148.0] 1432 | 1432  57 | 113 1609 | 1815  522 | 1295 574 | 620 211| 238 D-dimer VTE (mcg/mL FEU) [<0.65] 4.35 | 4.35 1.44 | 1.44 0.49 |0.49 >20 | >20 1.20 | 1.38 >20 | >20 ESR (mm/hr) [0-22] 78 | 78 Notavailable  6 | 12 Not available 35 | 74 Not available Procalcitonin(ng/ml) [0.00-0.49] 0.44 | 0.44 <0.1 | <0.1 <0.1 | <0.1 5.23 | 5.23 Notavailable 0.34 | 0.34 IL-6 (pg/ml) [<5.0] Not available 7 | 7 <5 | <5Not available Inconclusive Not available Immunomonitoring (initialrecoded value | lowest recored value) WBC (K/μL) [4.0-10.8] 2.3 | 2.36.9 | 5.2 4.4 | 3.2 17.7 | 7.9  3.4 | 2.4 17.4 | 5.9  Lymphocytes (K/μL)[0.6-4.9] 0.4 | 0.4 1.1 | 1.1 0.6 | 0.6 1.6 | 0.8 0.8 | 0.8 0.9 | 0.9CD3 (/μL) [510-2607] 636 1012  205 470 578 1373  CD3/CD4 (/μL)[302-1779] 415 562 129 329 386 982 CD3/CD8 (/μL) [101-951] 168 401  63126 169 336 Caspase-1 Activity (as determined by FAM-FLICA-caspaseassay) CD45 + CD3 + % [2.11-4.90]    22.38    26.41    16.85    18.88   14.30    21.16 CD45 + CD3 + CD4 + % [1.87-3.67]    17.61    24.93   16.77    10.66    12.30    21.22 CD45 + CD3 − % [3.77-16.6]    22.91   14.28    28.41    28.98    36.24    0.89 CD45 + CD3 + MFI[58.8-129.0] 124 112 110 119  98 154 CD45 + CD3 + CD4 + MFI [51.2-114.8]110 111 107 108  94 159 CD45 + CD3 − MFI [61.2-170.4] 156 112 135 164137  22 Patient 7 8 9 10 11 Transplant Info Organ type Kidney NA NALiver Liver Immunosuppression at presentation FK, NA MMF FK, MMF, FK,MMF, Prednisone Prednisone Steroids Potential COVID-19 Risk CategoriesAge > 50 Yes Yes Yes No Yes Gender M F F M F Race/ethnicity AA CaucasianAA Caucasian Other BMI  22  18  44  24  32 Comorbidities  5  3  >6  4  4Diabetes ✓ ✓ ✓ Obesity ✓ ✓ Hypertension ✓ ✓ ✓ Heart disease ✓ ✓ OtherGout, TB, Liver cirrhosis, COPD, AIH, AKI, HCV, Gout BK nephritis kidneyfailure, DVT, stroke, graft rejection hypercalcemia hyperlipidemiaCOVID-19 Course and Outcome Key symptoms Fever ✓ ✓ ✓ ✓ at time of Cough✓ ✓ ✓ ✓ presentation Dyspnea ✓ ✓ Myalgia/lethargy ✓ ✓ ✓ Diarrhea ✓Radiographic viral PNA detection ✓ ✓ ✓ ✓ ✓ Treatment Hydroxychloroquine✓ ✓ ✓ ✓ ✓ Azithromycin ✓ ✓ ✓ ✓ Outcome Hospitalization ✓ ✓ ✓ ✓ ✓Supplemental O₂ ✓ ✓ ✓ ✓ ✓ Intubation/ICU ✓ ✓ ✓ No discharge by d 14 ✓ ✓✓ ✓ ✓ Death   ✓□ Inflammatory Markers (initial recorded value | highestrecorded value) LDH (u/liter) [84-246] 257 | 443 162 | 190 388 | 488 352| 388 411 | 411 CRP (mg/L) [0.0-3.0]  63.3 | 107.0 3.4 | 5.4  96.2 |296.0  31.4 | 101.0 145.0 | 228.0 Ferritin (ng/mL) [5.0-148.0]  3254 |11230 1527 | 1527 259 | 439 2941 | 4585 2238 | 2754 D-dimer VTE (mcg/mLFEU) [<0.65] 14.35 | 14.35 2.43 | 2.43 1.92 | 3.56 5.71 | 5.71 11.55 |16.37 ESR (mm/hr) [0-22] 104 | 104 67 | 67  110 | >150 55 | 58  75 | 128Procalcitonin (ng/ml) [0.00-0.49] 46.2 | 46.2 0.12 | 0.12 <0.1 | 4.570.82 | 3.74 >150 | >150 IL-6 (pg/ml) [<5.0] 10 | 10 22 | 22 70 | 70 <5 |<5 <5 | <5 Immunomonitoring (initial recoded value | lowest recoredvalue) WBC (K/μL) [4.0-10.8] 7.4 | 3.7 2.7 | 2.7 3.7 | 3.7 4.2 | 4.2 8.0| 4.8 Lymphocytes (K/μL) [0.6-4.9] 1.2 | 0.9 0.9 | 0.6 0.9 | 0.4 0.2 |0.2 0.2 | 0.2 CD3 (/μL) [510-2607] 735 592 1071  210 106 CD3/CD4 (/μL)[302-1779] 611 404 668  42  56 CD3/CD8 (/μL) [101-951] 108 176 380 160 44 Caspase-1 Activity (as determined by FAM-FLICA-caspase assay) CD45 +CD3 + % [2.11-4.90]    13.79    20.21    19.68    28.44    18.36 CD45 +CD3 + CD4 + % [1.87-3.67]    10.95    15.63    21.54    30.83    16.77CD45 + CD3 − % [3.77-16.6]    12.32    2.86    3.68    6.23    28.99CD45 + CD3 + MFI [58.8-129.0] 110 128 156 189 117 CD45 + CD3 + CD4 + MFI[51.2-114.8] 105 115 164 205 100 CD45 + CD3 − MFI [61.2-170.4] 130  47 51  41 149 Items shaded in red denote test values or patientcharacteristics that either fall outside or at the boundary of thestandard reference range or are in a range that was highlighted asworrisome a recent NEJM paper (“Covid-19 and Kidney Transplantation”DOI: 10.1056/NEJMc2011117).

First, Applicants' patients also exhibited low T-cell counts andlymphopenia. In addition, Applicants see comorbidities with aninflammatory profile as especially high-risk for a morbid COVID-19course (Guo et al. Nat Med 2015; 21:677-687). Given this finding andstudies emphasizing the role of the inflammasome in coronaviruses (Shiet al. Cell Death Discov. 5, 101 (2019)), Applicants analyzed caspase-1activity in their patients. Critically, Applicants found a strongupregulation of caspase-1 activity in both T-cells and lymphocytes aswell as high LDH levels across their patients, suggesting pyroptosis asa cause of lymphopenia and a driver of heightened inflammation inCOVID-19 similar to SARS-CoV-1 (Yue et al. Cell Death Dis 9, 904(2018)). This insight has therapeutic implications beyondtransplantation as it implies upstream prevention of pyroptosis may bebeneficial.

Example 3: A Multicenter, Randomized Study to Evaluate the Safety andEfficacy of VX-765 in Improving Outcome in Patients with COVID-19Pneumonia

Methodology This study is conducted as a randomized safety, tolerabilityand efficacy study. Study duration is approximately 4 weeks. VX-765 isadministered daily up to 15 days. The study is conducted on anin-patient basis with assessments performed at screening and dailythereafter up to 15 days and at 28 days (study termination).

Study Duration Each subject participates in the study for 4 weeks. StudyCenters This is a multi-center trial with approximately 10 sites.

Objectives The purpose of this study is to evaluate the safety,tolerability and efficacy of VX-765 in subjects with Covid-19 infection.

Primary Safety and Tolerability Objective:

To determine the safety and tolerability of daily VX-765 for up to 15days in subjects with Covid-19 infection as determined by Adverse Eventsand Serious Adverse Events.

Primary Efficacy Objectives:

To determine the efficacy of VX-765 in improving outcome from Covid-19infection as determined by a 7-point ordinal disease severity scaledisease severity at 15 days.

Secondary Efficacy Objectives:

To determine the effect of VX-765 on outcome from Covid-19 infection asdetermined by improvement in the 7-point ordinal disease severity at 28days. Duration of mechanical ventilation, duration of hospitalization,oxygenation status, ventilator use time from treatment initiation todeath and virologic measures is assessed out to 28 days. Otherassessments include improvement in markers of pyroptosis such aslymphopenia, T cell counts, LDH levels, Caspase 1 activity levels andother secondary immune markers as read out parameters and/or secondaryend points.

Number of Subjects Approximately 80 subjects are entered into the studyStudy Description Multicenter study of daily VX-765 for up to 15 daysStudy Population Hospitalized patients with Covid-19 infection StudyDrug Administration Oral Inclusion/Exelusion Criteria The studypopulation is defined as subjects who meet the following criteria:

Inclusion Criteria:

Subject (or legally authorized representative) provides written informedconsent prior to initiation of any study procedures

Understands and agrees to comply with planned study procedures

Agrees to the collection of nasopharyngeal swabs and venous blood perprotocol

Male or non-pregnant female adult ≥18 years of age at time of enrollment

Laboratory confirmed SARS-CoV-2 infection as determined by PCR, or othercommercial or public health assay in any specimen <72 hours prior torandomization

Hospitalized patients with illness of any duration, and at least one ofthe following:

Clinical assessment (presence of crackles/rales) AND SpO2≤94% on roomair OR

Acute respiratory failure requiring mechanical ventilation and/orsupplemental oxygen OR

Radiographic infiltrates by imaging (CT, CXR)

Women of childbearing age must agree to use contraception for theduration of the study

Exclusion Criteria

Pregnant or breast feeding

Uncontrolled, clinically significant heart diseases such as arrhythmias,angina or uncompensated congestive heart failure

Non-hospitalized patients

Study Product, Dose, Route, Regimen VX-765 is administered in oral formthree times a day for up to 15 days.

The first group of subjects is dosed with 300 mg VX-765.

Study Endpoints Safety and Tolerability Endpoints:

Adverse Events and Serious Adverse Events out to 28 days, as evaluatedwith subject report, clinical assessment and vital signs, chemicalchemistry and electrocardiograms (EKG).

Primary Efficacy Endpoint:

The primary efficacy endpoint is the severity rating on the following7-point ordinal disease severity scale at 15 days.

Not hospitalized, no limitations on activities

Not hospitalized, limitation on activities

Hospitalized, not requiring supplemental oxygen

Hospitalized, requiring supplemental oxygen

Hospitalized, on non-invasive ventilation or high flow oxygen devices

Hospitalized, on invasive mechanical ventilation or ECMO

Death

Secondary Efficacy Endpoints:

The severity rating on the 7-point ordinal disease severity scale atdays 3, 6, 9, 12, and 28

Time to discharge or to a National Early Warning Score (NEWS) of <2maintained for 24 hours, whichever occurs first (at 3, 6, 9, 12, 15, and28 days)

Change from baseline in NEWS at days 3, 6, 9, 12, 15, and 28

Number of oxygen free days over 28 days

Incidence and duration of new oxygen use over 28 days

New mechanical ventilation over 28 days

Ventilator free days over 28 days

Duration of hospitalization

SARS-CoV-2 in nasopharyngeal sample at days 3, 6, 9, 12, 15, and 28

Quantitative SARS-CoV-2 virus in blood at days 3, 6, 9, and 12

Statistical Considerations Study Populations:

Safety Population: All enrolled subjects who have received at least 1dose of study drug

Efficacy Evaluable Population: All subjects in the Safety Population whocomplete the study with no major protocol violations

Analysis Methods:

Descriptive summary statistics is presented for the safety andtolerability endpoints overall. Continuous variables are summarizedusing the number of observations, mean, standard deviation, median,minimum, and maximum. Categorical variables are summarized usingfrequency counts and percentages. Summary statistics are provided forStage 1. Inferential statistics are performed in Stage 2.

Stage 2 Primary Efficacy Endpoint Analysis:

The primary efficacy endpoint analysis is a treatment group comparisonof the patients' day 15 7-point ordinal disease severity scale using aCochran-Mantel-Haenszel (CMH) row mean score difference test controllingfor the baseline score. The primary efficacy analysis compares thepooled Stage 2 placebo groups to the pooled Stage 2 VX-765 groups at thetwo-sided 0.05 Type I error rate.

Sample Size Justification:

For Stage 1, the sample size of 4 subjects per group (2 on VX-765, 2 onplacebo) is designed to provide an initial assessment of safety andtolerability for the initial doses of VX-765. The Stage 2 sample size of28 patients in a VX-765 treatment group (i.e., the 2 VX-765 treatmentgroups combined) provides a 95% probability that an AE with anunderlying rate of 10% occurs in these patients. Additionally, in Stage2, a sample size of 28 patients per group (i.e., pooling the Stage 2placebo groups and the VX-765 groups) provides approximately 80% powerto detect a treatment difference at the 2-sided 0.05 Type I error ratebased on a using a CMH row mean score difference test controlling forthe baseline score.

This assumes the following day 15 distribution for the seven-categorydisease severity score.

Distribution of 7-point Ordinal Disease Severity Scale at 15 Days forPower Calculations

7-Point Ordinal Disease Severity Scale 1 2 3 4 5 6 7 VX-765 30% 30% 20%13% 1% 1%  5% Placebo 10% 20% 20% 20% 5% 5% 20%

Safety and Tolerability Objectives

To determine the safety and tolerability of VX-765 for up to 15 days insubjects with Covid-19 infection as determined by Adverse Events andSerious Adverse Events

Safety and Tolerability Endpoints

Adverse Events and Serious Adverse Events out to 28 days, as evaluatedwith subject report, clinical assessment, vital signs, chemicalchemistry and electrocardiograms (EKG).

Primary Efficacy Objective:

To determine the efficacy of VX-765 in improving outcome from Covid-19infection as determined by the 7-point ordinal disease severity scale at15 days.

Primary Efficacy Endpoint:

The primary efficacy endpoint is the severity rating on the following7-point ordinal disease severity scale at 15 days.

Not hospitalized, no limitations on activities

Not hospitalized, limitation on activities

Hospitalized, not requiring supplemental oxygen

Hospitalized, requiring supplemental oxygen

Hospitalized, on non-invasive ventilation or high flow oxygen devices

Hospitalized, on invasive mechanical ventilation or ECMO

Death

Secondary Efficacy Objectives:

To determine the effect of VX-765 on outcome from Covid-19 infection asdetermined by improvement in the 7-point ordinal disease severity scaleat 28 days. Duration of mechanical ventilation, duration ofhospitalization, oxygenation status, ventilator use, time from treatmentinitiation to death and virologic measures is assessed out to 28 days.

Secondary Efficacy Endpoints:

The severity rating on the 7-point ordinal disease severity scale atdays 3, 6, 9, 12 and 28

Time to discharge or to a (NEWS) of <2 maintained for 24 hours,whichever occurs first (at 3, 6, 9, 12, 15 and 28 days)

Change from baseline in NEWS at days 3, 6, 9, 12, 15, and 28

The National Early Warning Score, (NEWS) has demonstrated the ability todiscriminate patients at risk of poor outcomes. This score is based onthe following 7 clinical parameters

Respiration rate

Oxygen saturation

Any supplemental oxygen

Temperature

Systolic blood pressure

Heart rate

Level of consciousness

Number of oxygen free days over 28 days

Incidence and duration of new oxygen use over 28 days

New mechanical ventilation over 28 days

Ventilator free days over 28 days

Duration of hospitalization

SARS-CoV-2 in nasopharyngeal sample at days 3, 6, 9, 12, 15, and 28

Quantitative SARS-CoV-2 virus in blood at days 3, 6, 9, and 12

During the 15-day dosing period, it is anticipated that no more than 2subjects out of 40 (5.0.0%) have an adverse event of grade 4 or 5 thatis at least possibly related to VX-765. Should there be more than 2subjects with an adverse event grade 4 or 5 that is at least possiblyrelated to VX-765 in patients receiving VX-765, the study is put on animmediate clinical hold.

In addition, individual safety stopping rules include:

Reaching dose-limiting tolerability (DLT) before clinical improvement

Having a non-DLT respiratory adverse event >grade 3 during studymedication inhalation that is at least possibly attributable to VX-765

Tolerability endpoints are referred to as the DLT and includewheezing/worsening of breathing.

The purpose of this randomized study is to obtain safety, tolerabilityand efficacy data in hospitalized patients with Covid-19 infectiontreated with VX-765 in addition to standard of care. The studydrug/placebo is delivered every 8 hours for up to 15 days.

The study is conducted in two stages. Stage 1 is single-blind (patientis blinded, but the physicians and sponsor is unblinded), whereas Stage2 is double-blind (patient and physician is blinded, sponsor isunblinded). In Stage 1, 24 patients is treated in groups of 4,randomized 1:1 to VX-765 or placebo and treated with escalating doses.The first group (n=4) is treated with VX-765 or placebo at 0.5 mg (in2.5 mls normal saline). The second group (n=4) is treated with VX-765 orplacebo at 1.0 mg (in 5 ml normal saline). Subsequent groups (each groupn=4) is treated with VX-765 or placebo at 2.0 mg (in 10 mls normalsaline), 3.0 mg (in 15 mls normal saline), 4.0 mg (in 20 mls normalsaline) or 5.0 mg (in 25 mls normal saline). In Stage 2, 28 patients israndomized 1:1 to VX-765 or placebo at the highest dose without anydrug-related adverse events, and another 28 patients is randomized 1:1to VX-765 or placebo at the dose just below the highest dose without anydrug-related adverse events.

Stage 1 Single-Blinded Cohort STAGE 1 Randomization 1:1 Cohort (n = 24)VX-765 + SOC* Placebo + SOC* 1 (n = 4) 10 mg/kg Placebo tablet 2 (n = 4)12.5 mg/kg Placebo tablet 3 (n = 4) 25 mg/kg Placebo tablet 4 (n = 4) 50mg/kg Placebo tablet 5 (n = 4) 75 mg/kg Placebo tablet 6 (n = 4) 100mg/kg Placebo tablet *SOC: Standard of Care Note: Based on a review ofthe safety and tolerability data of each dosing group, the DSMB has theoption of allowing an additional 4 patients to be randomized to a dosinggroup

Stage 2 Double-Blinded Cohort STAGE 2 Randomization 1:1^(a) Cohort (n =56) VX-765 Placebo 1 (n = 28) Highest dose at which no Highest dose atwhich no drug-related adverse events drug-related adverse eventsoccurred in Stage 1 occurred in Stage 1 2 (n = 28) Next highest doseNext highest dose ^(a)To balance the distribution of oxygen supportbetween the two groups as an indicator of severe respiratory failure,randomization in Stage 2 is stratified on the basis of respiratorysupport methods at the time of enrollment: no oxygen support with nasalduct or mask, or high flow oxygen, noninvasive ventilation, or invasiveventilation including ECMO.

Approximately 80 subjects are entered at up to 10 sites in the USA.Hospitalized patients with illness of any duration are assessed foreligibility on the basis of a positive reverse transcriptase polymerasechain reaction (RT-PCR) assay or other commercial or public health assayfor SARS-CoV-2 in a respiratory tract sample. Patients who test positivein any sample <72 hours prior to randomization are eligible. In additionto a positive SARS-CoV-2 test, patients are required to havecrackles/rales and an SpO2≤94% on room air, or respiratory failurerequiring supplemental oxygen/ventilation, or radiographic infiltratesby imaging (CT, CXR).

All eligible patients receive standard of care. Standard of care includesupplemental oxygen, noninvasive and invasive ventilation, antibiotics,vasopressors and ECMO.

In addition, patients receive study medication/placebo, administeredthree times a day for up to 15 days. The lowest dose group receives 10mg/kg orally. The highest dosing group receives 100 mg/kg orally.Medication is discontinued if the patient improves to the point wherethey can be discharged.

Patients are assessed daily for 15 days or until discharge, and again ondays 12, 15, and 28. All data is entered into an EDC daily.

Subjects who prematurely discontinue study drug for any reason are askedto have an Early Termination (ET) visit the day the last study drug doseis administered.

Schedule of Events

Time Point (Days) D 1 D 2 D 3 D 4 D 5 D 6 D 7 D 8 D 9 D 10 D 12 D 15 D28 I/E X Consent X Demo X Meds X X X X X X X X X X X X X PE X X ECG X XX X SpO2 X X X X X X X X X X X X X Labs X X X Pregnancy X Temp X X X X XX X HR X X X X X X X SBP X X X X X X X RR X X X X X X X X X X X X X LOCX X X X X X X CXR X X X CT* X X X CoV-2 N/P X X X X X X X CoV-2 X X X XX Blood AE X X X X X X X X X X X X X NEWS X X X X X X X 7-POS X X X X XX X IP/Placebo X X X X X X X X X X X X COVID-19 X X X X X X X X immunelabs I/E: Inclusion/Exclusion criteria; Demo: Demographics; PE: PhysicalExamination; Labs: CBC and chemistry; Pregnancy testing: For women ofchild-bearing potential, using urine pregnancy kit (UPT); SpO2:peripheral Oxygen Saturation; 7-POS: 7-Point Ordinal Disease SeverityScale; NEWS: National Early Warning Score; AE: Adverse Event; LOC: LevelOf Consciousness; CT*: CT scan is optional alternative to CXR; CXR:Chest X-Ray; CoV-2 N/P: Quantitative SARS-CoV-2 PCR from Nasopharyngealswab sample; CoV-2 Blood: Quantitative SARS-CoV-2 PCR from blood sample.IP: Investigational product or placebo administration. COVID-19 immunelabs include caspase-1, CBC with differential, T cell counts, LDH.

7-Point Ordinal Disease Severity Scale:

This is an assessment of the clinical status and it serves as theprimary endpoint. The scale is as follows:

Not hospitalized, no limitations on activities

Not hospitalized, limitation on activities

Hospitalized, not requiring supplemental oxygen

Hospitalized, requiring supplemental oxygen

Hospitalized, on non-invasive ventilation or high flow oxygen devices

Hospitalized, on invasive mechanical ventilation or ECMO

Death

The NEW Score has demonstrated the ability to discriminate patients atrisk of poor outcomes and is being used as an efficacy measure. Thisscore is based on 7 clinical parameters:

Respiration rate

Oxygen saturation

Any supplemental oxygen

Temperature

Systolic blood pressure

Heart rate

Level of consciousness

Quantitative Nasopharyngeal SARS-CoV-2 RNA testing (RT-PCR):

Nasopharyngeal or throat swabs are used. Any FDA approved or commercialtesting method is acceptable.

Quantitative blood SARS-CoV-2 RNA testing (RT-PCR):

Blood samples are used. Any FDA approved or commercial testing method isacceptable.

Study Population

Hospitalized patients with illness of any duration are assessed foreligibility on the basis of a positive reverse transcriptase polymerasechain reaction (RT-PCR) assay or other commercial or public health assayfor SARS-CoV-2 in a respiratory tract sample. Patients who test positivein any sample <72 hours prior to randomization are eligible.

Number of Subjects

Approximately 80 subjects are entered at up to 10 sites in the USA.

Selection Criteria

Hospitalized patients with illness of any duration are assessed foreligibility on the basis of a positive reverse transcriptase polymerasechain reaction (RT-PCR) assay or other commercial or public health assayfor SARS-CoV-2 in a respiratory tract sample. Patients who test positivein any sample <72 hours prior to randomization are eligible. In additionto a positive SARS-CoV-2 test, patients are required to havecrackles/rales and an SpO2≤94% on room air, or respiratory failurerequiring supplemental oxygen/ventilation, or radiographic infiltratesby imaging (CT, CXR).

Inclusion Criteria:

Subject (or legally authorized representative) provides written informedconsent prior to initiation of any study procedures.

Understands and agrees to comply with planned study procedures

Agrees to the collection of nasopharyngeal swabs and venous blood perprotocol.

Male or non-pregnant female adult ≥18 years of age at time ofenrollment.

Laboratory confirmed SARS-CoV-2 infection as determined by PCR, or othercommercial or public health assay in any specimen <72 hours prior torandomization.

Hospitalized patients with illness of any duration, and at least one ofthe following:

Clinical assessment (presence of crackles/rales) AND SpO2≤94% on roomair OR

Acute respiratory failure requiring mechanical ventilation and/orsupplemental oxygen OR

Radiographic infiltrates by imaging (CT, CXR)

Women of childbearing age must agree to use contraception for theduration of the study.

Exclusion Criteria:

Pregnant or breast feeding

Uncontrolled, clinically significant heart diseases such as arrhythmias,angina or uncompensated congestive heart failure

Non-hospitalized patients

Discontinuation Criteria and Early Termination Procedures

Subjects may withdraw voluntarily from participation in the study at anytime and for any reason. Subjects may also be withdrawn on the basis ofthe Investigator's clinical judgment.

When a subject withdraws or is withdrawn before completing the study,the date and reason for withdrawal are to be documented. Subjects whowithdraw or who are withdrawn prematurely are to attend an earlydiscontinuation visit, at which time they complete all assessments asoutlined in the Schedule of Events.

In the event that a subject is withdrawn prematurely due to an adverseevent or serious adverse event, the adverse event or serious adverseevent is followed until it resolves or stabilizes, or until it is judgedby the investigator to be no longer clinically significant.

Concomitant and Prohibited Medication

Hydroxychloroquine

Non-study drug anti-viral agents such as lopinavir, remdesivir,ritonavir, ribavirin or interferon-1β

Any other medication not approved for the treatment of SARS-CoV-2.

All study medication contains VX-765 in a 300 mg tablet, which may bebroken up depending on the dosage. The total dose administered is basedon the total weight administered to the patient.

Packaging and Labelling

Study medication is supplied in blinded, single-use syringes packaged inkits. Each kit contains either drug product tablets only or placebotablets only. Labelling clearly identifies that the tablets contain aninvestigational drug product but does not include any patient-specificinformation. The kit box labelling complies with regulatory requirementsfor investigational drug labelling and includes a pre-assignedindividual medication ID number for randomization assignment and storageconditions. Individual syringes within the kit are not labelled with themedication ID number and should not be removed from the kit until timeof use. Labelling includes a tamper-proof emergency use method forunblinding.

Storage, Dispensing and Reconciliation of Study Drug and Identity ofInvestigational Products

All study medication should be stored at room temperature untildispensed. Storage in hospital should be in a locked and secure locationaccessible only to site staff involved with this study.

If a hospital becomes aware that study medication has not been properlyhandled, the sponsor must be contacted immediately. In such an event,study medication should not be utilized until the sponsor providesfurther direction.

Neither the investigator nor any study personnel distributes any studymedication to any person not participating in this study. The studymedication is administered at the discretion and direction of theinvestigator in accordance with the conditions specified in thisprotocol. It is the investigator's responsibility to ensure thataccurate records of study medication issuance and return are maintained.

The sponsor is responsible for the tracking and accountability of studymedication dispensed to hospitals and informs hospitals how to return ordestroy study medication once it is no longer needed at the site.

Identification of Investigation Products

Product Name VX-765 Dosage form Tablet containing 300 mg VX-765Route/dosage Oral.

VX-765 doses are as follows:

10 mg/kg

12.5 mg/kg

25 mg/kg

50 mg/kg

75 mg/kg

100 mg/kg

(Placebo subjects receive same volume as treatment subjects at eachdosing level)

Dosing Instructions. Oral

Observations and Measurements

Subject informed consent must be obtained prior to conducting anystudy-related procedures. The informed consent can be signed by a nextof kin if necessary. The Principal Investigator at each center ensuresthat the subject is given full and adequate oral and written informationabout the nature, purpose, possible risk and benefit of the study.Subjects must also be notified that they are free to discontinue fromthe study at any time. The subject should be given the opportunity toask questions and allowed time to consider the information provided.

All assessments and procedures is completed according to the Schedule ofEvents.

Instructions to Subjects

VX-765 is administered by hospital staff. There are no specificinstructions to patients.

Warnings and Precautions

Pre-Existing Medical Conditions

All subjects enrolled in the study have SARS-CoV-2 infection andrespiratory symptoms.

Treatment Emergent Adverse events

A treatment-emergent adverse event (TEAE) is defined as any event notpresent prior to the initiation of the treatments or any event alreadypresent that worsens in either intensity or frequency following exposureto the treatments.

An adverse event is typically collected after signing the informedconsent form and could be related or unrelated to the study drug. A TEAEis for after the subject actually takes the study drug.

Separate summaries for adverse events that occur during treatment(summary of treatment emergent adverse events) is provided.

Laboratory Abnormalities

Clinical labs is performed by each hospital in their own laboratories.Labs to be drawn during the study include serum chemistries, ahematology panel and coagulation panel. A serum pregnancy test must beperformed, and the result must be negative prior to the entry of womenof child-bearing potential. COVID 19 immune labs (such as (caspase-1,CBC with differential, T cell counts, LDH) are also performed.

Clinical laboratory reports must be reviewed by a physician forout-of-range values within 12 hours of receipt. Out-of-range values isevaluated using the following notations:

NS: Not clinically significant

LE: Laboratory Error

PT: Subject abnormal; relates to the subject's usual state of health

CS: Clinically Significant. This value cannot be explained by any of theother flags.

By definition a lab value flagged as “CS” must be entered on the adverseclinical laboratory event page in the CRF. A laboratory test flagged“CS” must be repeated as soon as possible. The investigator may usehis/her own judgment as to whether the abnormal finding is sufficientreason to immediately withdraw the subject from the study.

If a laboratory value is considered to be serious and life-threateningand at least possibly attributable to the study drug, the subject shouldbe immediately discontinued from the study and appropriate therapystarted.

Adverse Event Assessment and Recording

All adverse events, exacerbations of concomitant illnesses, or eventsknown to be related to underlying disease processes or concomitantmedications are to be recorded on the CRF throughout the study. If apre-existing condition worsens on study, the date on which theexacerbation began should be recorded. Onset dates for studytreatment-related adverse events must be on or after the date of initialstudy treatment use. The need to record an adverse event on the CRF isnot dependent on whether the adverse event is associated with the use ofthe study medication. In order to avoid vague, ambiguous or colloquialexpressions, the adverse event should be recorded in standard medicalterminology.

Adverse event recording includes the date of onset, severity, duration,whether or not the study medication was discontinued because of theevent, the treatment given and the outcome. The investigator must alsoassess whether the event was related to the study medication, concurrentdrug therapy, underlying disease, a combination of these factors, or ifit is unknown. Subjects with an adverse event should be carefullyfollowed to determine outcome.

The investigator uses the National Cancer Institute (NCI) definitions tograde the severity of the event.

Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnosticobservations only; intervention not indicated

Grade 2: Moderate; minimal, local, or non-invasive interventionindicated; limiting age-appropriate ADL

Grade 3: Severe or medically significant but not immediatelylife-threatening; hospitalization or prolongation of hospitalizationindicated; disabling; limiting self-care ADL.

Grade 4: Life-threatening consequences; urgent intervention indicated

Grade 5: Death

The relationship or association of the study medication in causing orcontributing to the adverse event is characterized as remote, possibleor probable as defined below:

Not related: Evidence indicates no plausible direct relationship to thestudy medication

Remote: Suggests other conditions are reasonably likely to account forthe event including concurrent illness, progression or expression of thedisease state, or reaction to concurrent medication

Possible: Suggests that the association of the event with the studymedication is unknown; however, the adverse event is not reasonablysupported by other conditions

Probable: Suggests that a reasonable temporal sequence of the event withmedication administration exists and, based upon the investigator'sclinical experience, the association of the event with study medicationseems likely

Definite: Suggests that based upon the investigator's experience, theassociation of the event with the study medication seems very certain.

Procedures such as surgery should not be recorded as adverse events.However, the medical condition for which the procedure was performedshould be reported if it meets the definition of adverse event.

Reporting Requirements

Any adverse event, including both observed or volunteered problems,complaints, or symptoms that begins any time between the start of thefirst dose and within 30 days after the end of the last dose are to berecorded briefly on the appropriate CRF and in detail in the sourcedocuments. A check list of adverse events may not be used during thisstudy.

The following are specific definitions that are relevant to meeting yourreporting obligations and which are included in the FDA Regulations, 21CFR Part 312.32, and International Conference on Harmonization ofTechnical Requirements for Registration of Pharmaceuticals for Human Use(ICH) Guidelines:

Adverse Event: Any untoward medical occurrence in a subject administereda pharmaceutical product and which does not necessarily have to have acausal relationship with this treatment. An adverse event can be anyunfavorable sign (including an abnormal laboratory finding), symptom, ordisease temporally associated with the use of the investigational drug,whether or not considered related to the investigational drug.

Serious Adverse Event: An untoward event or reaction that at any dose:

results in death

is life-threatening

prolongs existing hospitalization

results in permanent or significant disability or incapacity

requires intervention to prevent permanent impairment/damage

Life-threatening: An event which a subject was at risk of death at thetime of event.

There is a distinction between the severity and the seriousness of anadverse event. Severe is a measurement of intensity, thus a severereaction is not necessarily a serious adverse event. For example, aheadache may be severe in intensity, but would not be serious unless itmet one of the criteria for serious adverse events listed previously.

Serious Adverse Events

Adverse events (AEs) and serious adverse events (SAEs) are collectedfrom the first dose of IP is administered until 28 days after the lastdose of IP. Medical occurrences that began prior to the start of studytreatment, but after obtaining informed consent were recorded on theMedical History/Current Medical Conditions CRF. The investigator or sitestaff is responsible for the detection and documentation of eventsmeeting the criteria and definition of an adverse event or seriousadverse event, as provided in the study protocol. However, any SAEsassessed as related to study participation (e.g., dosing, protocolmandated procedures, invasive tests, or change in existing therapy) orrelated to a concomitant medication is recorded from the time a subjectconsent to participate in the study up to and including any follow-upcontact.

In the event of an AE or SAE, it is the responsibility of theInvestigator to review all documentation (e.g., hospital progress notes,laboratory, and diagnostics reports) relative to the event and attemptto establish a diagnosis of the event based on signs, symptoms, andother clinical information. The diagnosis was to be documented as theadverse event or serious adverse event and not the individual'ssigns/symptoms. Once an Investigator becomes aware that a SAE hasoccurred in a study subject, they are′ to report the information within24 hrs and provide an assessment of causality.

Notification of Serious Adverse Events

Under IND regulations, 21 CFR Part 312.64, investigators are required tonotify the Sponsor promptly, within 24 hours of the sites' notificationof any SAEs, deaths, or life-threatening problems with theinvestigational drug. This regulation also requires that if the adverseevent is alarming, the investigator must notify the Sponsor immediately.

A Serious Adverse Event (SAE) is a significant hazard, contraindication,or precaution considered to be serious. This includes, but is notlimited to, blood dyscrasias, endocrine disturbances, hemorrhage fromany site, or severe skin disorder. Additional examples are intensivetreatment for allergic bronchospasm, blood dyscrasias or convulsions.

Subjects who experience a SAE must be given appropriate examinations andtreatment. The investigator must provide written information to thesponsor as soon as possible.

When an investigator is in doubt when to report an event, theinvestigator should err on the side of caution.

All appropriate SAEs is reported immediately to appropriate regulatoryauthorities by the Sponsor. A copy of all FDA reportable serious adverseevents is mailed to all investigators participating in ongoing clinicalstudies with the study medication in order to permit prompt notificationof the appropriate institutional review board (IRB).

Departure from Protocol for Emergency or Adverse Event

In medical emergencies, the investigator should use medical judgment andremove the subject from immediate hazard and decide whether the subjectmay continue in the study. The IRB should also be notified as to thetype of emergency and the course of action. The CRF for the subject mustdescribe the departure from the protocol and state the reason.

Safety Monitoring

A Data Safety and Monitoring Board (DSMB) has been established tomonitor the safety of the subjects during the study. The DSMB includesmembers who are independent of this study and its Sponsor Team withrelevant clinical expertise, including a good understanding of thesafety of medications for respiratory illnesses. These members include astatistician, an infectious disease specialist and/or a pulmonologist.The methodology and the operating procedures for the safety reviews isdeveloped by the infectious disease specialist and/or pulmonologist incollaboration with the sponsor and is documented in the DSMB Charter.The DSMB is not blinded. They review all SAEs and determine whether thestudy can proceed or whether protocol modifications are required.

Stopping Rules

During the 15-day dosing period, it is anticipated that no more than 2subjects out of 40 (5.0%) have an adverse event of grade 4 or 5 that isat least possibly related to VX-765. Should there be more than 2subjects with an adverse event grade 4 or 5 that is at least possiblyrelated to VX-765 in the cohort of subjects, the study is put on animmediate clinical hold.

In addition, individual safety patient study stopping rules include:

Reaching dose-limiting tolerability (DLT) before clinical improvement

Having a non-DLT respiratory adverse event >grade 3 within 24 hours oftaking study medication that is at least possibly attributable to VX-765

Follow-Up and Final Reports

The investigator shall provide an accurate final report within 1 monthafter completion, termination or discontinuation of the study. The finalreport may not precede completion of monitoring relevant CRFs.

Regulatory Aspects

Neither the investigator shall modify this protocol without firstobtaining concurrence of the other in writing. All changes must besubmitted to the IRB. Protocol modifications which impact subjectsafety, or the validity of the study must be approved by the IRB andsubmitted to the FDA before implementation. In the case of a medicalemergency to increase safety of subjects, a change may be made afterdiscussion with the sponsor. In these instances, the IRB and FDA isnotified as soon as possible.

The following populations are considered for statistical analyses.

Safety Population: The Safety Population consists of all patients whoreceive at least one dose of study drug.

Efficacy Evaluable Population: The Efficacy Evaluable Populationincludes all patients in the Safety Population who complete the dosingperiod with no major protocol violations.

Analysis Methods

General Methods

Details of the statistical analysis methodology is provided in aseparate statistical analysis plan (SAP), which is finalized prior tothe interim analysis being performed.

Continuous variables are summarized using the number of observations,number of observations above the limit of quantification (ifapplicable), mean, standard deviation (SD) median, and range.Categorical variables is summarized using frequency counts andpercentages.

Analysis of Subject Disposition, History, and Baseline Characteristics

Subject disposition, including analysis population allocation, subjectsenrolled, completed each period, discontinued, and primary reason fordiscontinuation, is summarized using frequency and percentage.

Protocol deviations is summarized using frequency and percentage.

Medical history data and prior and concomitant medications is summarizedusing frequency and percentage.

Subjects' age, height, weight, and baseline disease characteristics issummarized using descriptive statistics. Gender, race, and othercategorical variables is provided using frequency and percentage.

Safety Analyses

All safety analyses are performed on the Safety Population. The safetydata is presented in individual listings and summary tables summarizingresults by cohort and treatment group.

Adverse Events

AEs are coded according to the latest version of the Medical Dictionaryfor Regulatory Activities (MedDRA). The intensity/severity of AEs isgraded according to NCI CTCAE.

TEAEs, AEs leading to study treatment discontinuation, AEs leading todose interruption, AEs related to study medication, SAEs, and AEsleading to death are summarized by system organ class, preferred term,and study period. A summary of AEs that are CTCAE Grade 3 or higher, aswell as the most frequent preferred terms, is provided.

If a subject experiences the same preferred term multiple times within aperiod, then the event is counted only once within the period and bygreatest severity.

Descriptive statistics are used to summarize the safety data both bytreatment group and overall.

Clinical Laboratory Values

All laboratory test results are summarized by period together with thechange from baseline. The frequency distribution for low/normal/high ornormal/abnormal is summarized as well. The denominators for calculatingthe percentages are based on the number of subjects with non-missingvalues in the Safety Population.

Vital sign results are summarized by period, together with the changefrom baseline.

Summaries of physical examinations present frequency distribution ofabnormal findings by body system and period. The denominators forcalculating the percentages are based on the number of subjectsevaluated for a particular body system of each dose level in the SafetyPopulation.

Electrocardiogram (EKG) findings are classified as normal vs abnormal.The number and percentage of each category are summarized usingfrequency table for each period. The denominators for calculating thepercentages are based on the number of subjects with non-missing valuesin each period.

Tolerability is measured by DLT endpoints.

The frequency of occurrence for each tolerability endpoint includingwheezing and increasing difficulty in breathing is summarized by cohortand treatment group. Percentages are based on the number of subjects ineach dosing level in the Safety Population.

Efficacy Analyses

Primary Efficacy Endpoint Analyses

Summary statistics are provided for Stage 1, in Stage 2 inferentialstatistics are also performed.

Stage 2 Primary Efficacy Endpoint Analyses

The primary efficacy endpoint analysis is a treatment group comparisonof the patients' day 15 7-point ordinal disease severity scale using aCochran-Mantel-Haenszel (CMH) row mean score difference test controllingfor the baseline score. The primary efficacy analysis compares thepooled Stage 2 placebo groups to the pooled Stage 2 VX-765 groups at thetwo-sided 0.05 Type I error rate.

The primary efficacy analysis is performed on the Safety population.

Sensitivity analyses for the primary efficacy endpoint includingcomparing the individual VX-765 groups to placebo are specified.

Stage 2: Secondary Efficacy Endpoint Analyses

The Stage 2 secondary efficacy endpoint analysis methods are specified.

Handling of Missing Data

The approaches to handling missing data are specified.

Sample Size/Power Considerations

For Stage 1, the sample size of 4 subjects per group (2 on VX-765, 2 onplacebo) is designed to provide an initial assessment of safety andtolerability for the initial doses of VX-765. The Stage 2 sample size of28 patients in an VX-765 treatment group (i.e., the 2 VX-765 treatmentgroups combined) provides a 95% probability that an AE with anunderlying rate of 10% occurs in these patients. Additionally, in Stage2, a sample size of 28 patients per group (i.e., pooling the Stage 2placebo groups and the VX-765 groups) provides approximately 80% powerto detect a treatment difference at the 2-sided 0.05 Type I error ratebased on a using a CMH row mean score difference test controlling forthe baseline score. This assumes the following day 15 distribution forthe seven-category disease severity score. Power was estimated based onthe distribution of scores presented using the Mantel-Haenszel meanscore statistic with modified ridit scoring.

Distribution of 7-point Ordinal Disease Severity Scale at 15 Days forPower Calculations

7-Point Ordinal Disease Severity Scale 1 2 3 4 5 6 7 VX-765 30% 30% 20%13% 1% 1%  5% Placebo 10% 20% 20% 20% 5% 5% 20%

This study has an estimated maximum duration of up to 4 weeks for eachsubject. The study duration from first subject enrolled to last subjectout is expected to be 6 months.

The Principal Investigator(s) at each center ensures that the subject isgiven full and adequate oral and written information about the nature,purpose, possible risk and benefit of the study. Subjects must also benotified that they are free to discontinue from the study at any time.The subject should be given the opportunity to ask questions and allowedtime to consider the information provided.

The subject's informed consent must be obtained before conducting anystudy procedures.

During the study, a dedicated Clinical Research Associate (CRA) hasregular contacts with the investigational site, for the following:

Provide information and support to the investigator(s)

Confirm that facilities remain acceptable

Confirm that the investigational team is adhering to the protocol, thatdata are being accurately recorded in the eCRFs, and thatinvestigational product accountability checks are being performed

Perform source data verification. This includes a comparison of the datain the eCRFs with the subject's medical records at the hospital orpractice, and other records relevant to the study. This requires accessto all original records for each subject (e.g. clinic charts) asdescribed in the study monitoring plan.

The CRA is available over the course of the study if the investigator(s)or other staff needs information or advice. Remote and centralmonitoring visits are conducted until it is possible to perform on-sitevisits.

Audits and Inspections

Authorized representatives of a regulatory authority, an IndependentEthics Committee (IEC) or an Institutional Review Board (IRB) may visitthe site to perform audits or inspections, including source dataverification. The purpose of an audit or inspection is to systematicallyand independently examine all study-related activities and documents todetermine whether these activities were conducted, and data wererecorded, analyzed, and accurately reported according to the protocol,Good Clinical Practice (GCP) guidelines of the International Conferenceon Harmonization (ICH), and any applicable regulatory requirements.

Ethics Committee Review

The final study protocol, including the final version of the InformedConsent Form, must be approved or given a favorable opinion in writingby an IRB or IEC as appropriate.

The Principal Investigator is responsible for informing the IRB or IECof any amendment to the protocol in accordance with local requirements.In addition, the IRB or IEC must approve all advertising used to recruitsubjects for the study. The protocol must be re-approved by the IRB orIEC upon receipt of amendments and annually, as local regulationsrequire.

The Principal Investigator is also responsible for providing the IRBwith reports of any reportable serious adverse drug reactions from anyother study conducted with the investigational product.

Progress reports and notifications of serious adverse drug reactions areprovided to the IRB or IEC according to local regulations andguidelines.

The study is performed in accordance with ethical principles that havetheir origin in the Declaration of Helsinki and are consistent withICH/GCP, applicable regulatory requirements on Bioethics.

Any research information obtained about the subject in this study iskept confidential. A subject is not identified by name, only by his/herinitials. The subject's name or any identifying information does notappear in any reports published as a result of this study.

However, information obtained from individual subject's participation inthe study may be disclosed with his/her consent to the health careproviders for the purpose of obtaining appropriate medical care. Thesubject's medical records/charts, tests with his/her name on them may bemade available to the appropriate contract research organization (CRO),its potential eventual partners, and any other regulatory authorities.This is for the purpose of verifying information obtained for thisstudy. Confidentiality is maintained throughout the study within thelimits of the law.

A subject's name is not given to anyone except the researchersconducting the study, who have pledged an oath of confidentiality. Allidentifying information is kept behind locked doors, under thesupervision of the study Investigator and is not transferred outside ofthe investigator site.

A subject may take away his/her permission to collect, use and shareinformation about him/her at any time. If this situation occurs, thesubject is not able to remain in the study. No new information thatidentifies the subject is gathered after that date. However, theinformation about the subject that has already been gathered andtransferred may still be used and given to others as described above inorder to preserve the scientific integrity and quality of the study.

The site maintains records of study treatment delivered to the studysite; the inventory at the site; the administration to each subject; andthe return of materials for storage or disposal. These records shouldinclude dates, quantities, batch/serial numbers, expiration dates,in-clinic temperature log, and unique code numbers assigned to theproduct and study subjects.

Investigator maintains records that document adequately that thesubjects were provided with the correct study drug and reconciles theproducts received from the drug dispensing center. Investigationalproduct is not returned until accountability has been fully monitored.

Modifications to the protocol are only possible by approved protocolamendments. All protocol amendments are approved by the appropriateregulatory authorities as well as each institutional review board priorto implementation. The Investigator must not implement any deviationsfrom, or changes to the protocol, except where it is necessary toeliminate an immediate hazard to the study subject.

The protocol must be read thoroughly, and the instructions followedexactly. Any deviation to the protocol has to be reported as soon aspossible to Clinical Research Associate/designee. The governingreporting guidelines for protocol deviations must be adhered to by theInvestigator.

This study may be prematurely terminated, if in the opinion of theInvestigator, there is sufficient reasonable cause. Written notificationdocumenting the reason for study termination is provided to theInvestigator by the terminating party. Circumstances that may warranttermination include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk tosubjects.

Failure to enroll subjects at an acceptable rate.

Insufficient adherence to protocol requirements.

Insufficient complete and/or evaluable data.

Plans to modify, suspend, or discontinue the development of VX-765.

Should the study be closed prematurely, all study materials must bereturned.

Inspection of Records

The Investigator agrees to allow inspection of the drug storage area,study drug stocks, drug accountability records, subject charts and studysource documents, and other records relative to study conduct.

All data relating to study procedures is entered on to eCRFs. All formsmust be completed electronically. All requested information must beentered in the eCRF. Sites are trained on the eCRF Completion Guidelineswhich provided instructions on how to enter the required data in theElectronic Data Capture (EDC) system.

eCRFs are completed for each subject. It is the Investigator'sresponsibility to ensure the accuracy, completeness, and timeliness ofthe data entered in each subject's eCRF. Source documentation supportingthe eCRF data should indicate the subject's participation in the studyand document the dates and details of study procedures, adverse events,and subject status.

The Investigator, or designee, should complete the eCRF as soon aspossible after information is collected, preferably on the same day thata subject is seen for an examination, treatment, or any other studyprocedure. Any outstanding entries must be entered immediately after thefinal examination. An explanation should be given for all missing data.

Study records and source documents must be preserved for the longer of(i) two (2) years following completion of the study; or (ii) two (2)years following the termination or withdrawal of the Investigational NewDrug application under which this study was conducted; or (iii) theperiod required by local, state, and federal laws, regulations and FDAGuidance.

The Investigator agrees to comply with all applicable federal, state,and local laws and regulations relating to the privacy of subject healthinformation, including, but not limited to, the Standards forIndividually Identifiable Health Information, 45 CFR, Parts 160 and 164(the Health Insurance Portability Accountability Act of 1996 [HIPAA]Privacy Regulation). The Investigator shall ensure that study subjectsauthorize the use and disclosure of protected health information inaccordance with HIPAA Privacy Regulation.

Participating sites, the study database, and study documentationincluding subject medical records may be subject to a quality assuranceaudit during the course of the study. In addition, inspections may beconducted by regulatory bodies at their discretion.

Laboratory Tests

Hematology/Immunology Serum Chemistry Urinalysis White Blood Cell (WBC)Count Albumin Appearance Red Blood Cell (RBC) Count AlanineAminotransferase (ALT) pH Hemoglobin (Hb) Alkaline Phosphatase (ALP)Protein Hematocrit (Hct) Aspartate Aminotransferase (AST) Glucose MeanCorpuscular Volume (MCV) Blood Urea Nitrogen (BUN)/Creatinine KetoneBodies Red Blood Cell Distribution Width Ratio Indicators of Blood andWBCs (RDW) Calcium Specific Gravity Platelet Count Carbon DioxideUrobilinogen ESR sedation rate Chloride Differential - absolute andpercent Creatinine of: Glucose Neutrophils Magnesium LymphocytesPotassium Monocytes Sodium Eosinophils Total Bilirubin Basophils TotalProtein COVID-19 immune panel Urea Nitrogen (Caspase-1 levels in T cellsand Lactate Dehydrogenase (LDH) lymphocytes, and absolute countsCreatine Kinase, Total and % of CD3+ T cells, CD4+ T cells, CD8+ Tcells) Pregnancy tests A serum pregnancy test is performed on all femalesubjects of child-bearing potential at the screening visit. UrinePregnancy Test (on site): Urine human chorionic gonadotropin (HCG)(pre-menopausal females only)

Example 4: Safety, Tolerability, and Treatment Effect of Belnacasan inPatients with COVID-19: A Proof of Concept Randomized, Double-Blind,Placebo-Controlled Trial of Orally Administered Belnacasan Tablets forthe Treatment of Mild to Moderate COVID-19 Abbreviations

9-POS 9-Point Ordinal Disease Severity Scale

ADL Activities of daily living

AE Adverse event

ALC Absolute lymphocyte count

ALP Alkaline phosphatase

ALT Alanine aminotransferase

AST Aspartate aminotransferase

BMI Body mass index

CFR Code of Federal Regulations

CRF Case report form

CRP C-reactive protein

CS Clinically significant

CT Computed tomography

COVID-19 Coronavirus disease 2019

CTCAE Common terminology criteria for adverse events

CXR Chest x-ray

DLT Dose limiting toxicity

DSMB Data and safety monitoring board

ECMO Extracorporeal membrane oxygenation

EDC Electronic data capture

EKG Electrocardiogram

ET Early termination

FDA Food and Drug Administration

G-CSF Granulocyte colony-stimulating factor

GCP Good Clinical Practice

GGT Gamma-glutamyl transferase

HIPAA Health Insurance Portability and Accountability Act

HR Heart rate

I/E Inclusion/Exclusion

ICH International Council for Harmonization

IDS Investigational Drug Services

IND Investigational New Drug

IP Investigational product

IRB Institutional review board

LAR Legally authorized representative

LDH Lactate dehydrogenase

LOC Level of consciousness

NCS Not clinically significant

PE Physical examination

P-gp P-glycoprotein

QID Four times a day

RP MedStar Health Research Pharmacy

RR Respiratory rate

RT-PCR Reverse transcriptase polymerase chain reaction

SAE Serious adverse events

SAP Statistical analysis plan

SARS Severe acute respiratory syndrome

SBP Systolic blood pressure

SOC Standard of care

SpO2 Peripheral capillary oxygen saturation

TID Three times a day

TNF-α Tumor necrosis factor alpha

WHC Washington Hospital Center

WHO World Health Organization

Safety, tolerability, and treatment effect of belnacasan in patientswith COVID-19: A phase 2, proof of concept randomized, double-blind,placebo-controlled trial of orally administered belnacasan tablets forthe treatment of mild to moderate COVID-19

Study Phase. 2/Proof of Concept

Methodology. This study is conducted as a proof of concept safety,tolerability and treatment effect Phase 2 trial in subjects withconfirmed SARS-CoV-2 infection and mild to moderate COVID-19.

In this hypothesis generating, randomized, double-blind,placebo-controlled trial, 24 subjects are given 900 mg TID of belnacasanand 24 subjects are given a placebo TID. Belnacasan or placebo are givenfor 28 days and assessments of safety, tolerability, and treatmenteffect is performed for 60 days. Subjects in both treatment arms arealso treated with the background standard of care that is at that timeappropriate and commensurate with their COVID-19 disease severity.

Subjects use a diary to record study drug intake as well as symptoms andactivity levels; assess physical parameters using a study-providedthermometer and pulse oximeter; be assessed at regular intervals viatelemedical and in-person follow-up clinic visits; and give blood forlaboratory tests. A Data Safety & Monitoring Board (DSMB) is engagedthroughout for data monitoring and safety evaluation.

Study Duration. Subjects are in the study for 60 days. The study lasts3-6 months.

Study Centers. This study is conducted at Washington Hospital Center(WHC) and/or other MedStar hospital and/or critical or clinical caresites.

Objectives. The purpose of this trial is to assess the safety,tolerability and treatment effect of the orally administered Caspase-1inhibitor, belnacasan, for the treatment of patients with mild tomoderate COVID-19 and to generate hypotheses for future trials.

Primary Objective: To determine the safety and tolerability ofbelnacasan (VX-765/RVT-201/MSR-001) administered orally for 28 days insubjects with mild to moderate COVID-19 as determined by Adverse Eventsand Serious Adverse Events.

Secondary Objective: To generate data on the treatment effect ofbelnacasan (VX-765/RVT-201/MSR-001) administered orally for 28 days insubjects with mild to moderate COVID-19 as determined by its effect sizeon outcomes in five areas:

Clinical recovery and resolution of COVID-19 symptoms

Physical functions and parameters relevant to COVID-19

COVID-19 related deterioration and mortality

WHO 9-point ordinal scale for clinical improvement

Surrogate markers of COVID-19-related inflammation and organ involvement

The hypothesized end result of treatment with belnacasan would be toreduce the symptomatic and clinical burden, length of disease course,and disease progression of COVID-19.

Number of Subjects. The intent is to enroll 48 subjects to participatein a 1:1 allocation in this randomized, double-blind, placebo-controlledsafety, tolerability and treatment effect trial.

Study Description. A Phase 2, proof of concept randomized, double-blind,placebo-controlled trial of orally administered belnacasan tablets forthe treatment of mild to moderate COVID-19. Belnacasan or placebo isadministered for 28 days and follow-up occurs for 60 days, alongsidebackground standard of care.

Study Population. Subjects with an RT-PCR assay confirmed SARS-CoV-2infection and mild to moderate COVID-19 at time of enrollment.

Study Drug Administration. Oral, three times per day as per protocol.

Inclusion/Exclusion Criteria. The study population is defined assubjects who meet the following criteria:

Inclusion Criteria:

Subject (or legally authorized representative) provides written informedconsent prior to the initiation of any study procedures.

Subject understands and agrees to comply with planned study procedures,including using the diary.

Subject agrees to the collection of nasopharyngeal swabs and venousblood per protocol.

Subject is male or non-pregnant female adult ≥18 years of age at time ofconsent.

Women with a history of menstruation must agree to use two methods ofcontraception, at least one of which is highly effective, for theduration of the study as well as to undergo additional pregnancy testingduring the study.

Subject has a laboratory confirmed SARS-CoV-2 infection as determined byRT-PCR assay prior to enrollment.

Subject has evidence of either mild or moderate COVID-19 illness of lessthan 7 days from first onset, with minimal baseline symptom severitybased on patient-reported FDA scoring system defined as follows:

Subject presents with at least two common symptoms of COVID-19 from thefollowing list: stuffy or runny nose, sore throat, cough, low energy ortiredness, muscle or body ache, headache, chills or shivering, feelinghot or feverish, nausea, vomiting, diarrhea, shortness of breath withexertion (without supplemental oxygen requirement) with a score of 2 orhigher; impairment in sense of smell or taste with a score of 1 orhigher OR

Subject presents with any (i.e., at least one) symptom of COVID-19 asdefined above AND clinical evidence of moderate COVID-19 as defined byFDA guidance for industry (such as respiratory rate >20 breaths perminute, heart rate >90 beats per minute, with oxygen saturation >93% onroom air at sea level).

Subject presents with high-risk for COVID-19-related inflammationdetermined by at least one comorbidity, including obesity, diabetes,hypertension, stable heart disease, respiratory disease, or non-severefatty liver disease and/or age >60 years.

Subject's overall health condition is deemed as suitable to fully andsafely participate in this trial as determined by the Investigator.

Exclusion Criteria

Any clinical signs indicative of severe or critical COVID-19 as definedby FDA guidance for Industry at the time, including SpO2<93% and/oroxygen requirement.

Hospitalization for COVID-19, or consideration thereof

ICU level of care and/or non-mechanical/mechanical ventilation and/oroxygen supplementation at time of enrollment.

Pregnant or breast-feeding subjects.

Subjects who cannot swallow tablets.

History of any pre-existing organ impairment, such as:

Severe kidney disease (known or estimated GFR <30 mL/minute) or ondialysis.

Uncontrolled, clinically significant heart diseases such as arrhythmias,angina or heart failure as defined by AHA/ACC Grade C and D.

Chronic respiratory disease requiring supplemental oxygen.

Moderate and severe hepatic impairment as defined by Child-Pugh scoringClass B and Class C

Elevated liver function tests (determined by ALT, AST, GGT, or ALP >2×upper limit of normal, and total Bilirubin >upper limit of normal).

History of malignancy or immunodeficiency within the prior 5 years.

Acute respiratory illness other than COVID-19.

Active bacterial, viral or fungal infection (including HIV, hepatitis B,hepatitis C).

While dosed with the IP, the taking of prohibited concomitant medicationor the ingestion of food that interferes with the IP, including:

Non-COVID19-related anti-viral medication such as lopinavir, ritonavir,ribavirin or interferon-1β.

Systemically administered immunosuppressive and anti-inflammatoryagents, other than background standard of care for COVID-19 at the time.

Drugs and foods that are potent inhibitors or inducers of CYP3A4 and/orP-gp, as listed in FDA “Drug Development and Drug Interactions: Table ofSubstrates, Inhibitors and Inducers”, including (including herbalmedications such as St. John's Wort) within 30 days or 5 half-lives(whichever is longer) prior to the first dose of study drug.

Any other diseases or medical conditions or concomitant medications thatare deemed as not compatible or appropriate for the subject's ability tofully and safely participate in this trial as determined by theInvestigator.

Study Product, Dose, Route, Regimen. 48 subjects participate in thisrandomized, double-blind, placebo-controlled safety, tolerability andtreatment effect trial. In a 1:1 allocation, 24 subjects receive 900 mg(three tablets each containing 300 mg) TID of orally administeredbelnacasan and 24 subjects receive placebo tablets (three placebotablets) TID, whereby randomization is determined via a randomizationtable.

Study Endpoints. Primary Endpoint:

Incidence of Adverse Events and Serious Adverse Events assessed out today 60.

Secondary Endpoints:

Secondary endpoints relate to generating data on the treatment effect ofbelnacasan as determined by its effect size on outcomes in five areas:

1. Clinical Recovery and Resolution of COVID-19 Symptoms

1a. Sustained recovery and resolution rates of common COVID-19 symptoms:

(i.e., stuffy or runny nose, sore throat, cough, low energy ortiredness, muscle or body ache, headache, chills or shivering, feelinghot or feverish, nausea, vomiting, diarrhea, shortness of breath withexertion; impairment in sense of smell or taste)

Proportion of subjects in treatment group versus placebo group,respectively, who per symptom questionnaire on days 4, 7, 10, 14, 21,28, 42, 60 post randomization have achieved for two consecutive days:

scores not higher than 0 for all symptoms

scores not higher than 1 for all symptoms

scores not higher than 0 for all symptoms other than impairment of tasteor smell

scores not higher than 1 for all symptoms other than impairment of tasteor smell

1b. Sustained improvement of global impression rates: Proportion ofsubjects in treatment group versus placebo group, respectively, who persymptom questionnaire on days 4, 7, 10, 14, 21, 28, 42, 60 postrandomization have answered for two consecutive days

“Yes” to “In the past 24 hours, have you returned to your usual health(before your COVID-19 illness)?”

“Yes” to “In the past 24 hours, have you returned to your usualactivities (before your COVID-19 illness)?”

“None” to “In the past 24 hours, what was the severity of your overallCOVID-19 related symptoms at their worst?”

“Mild” to “In the past 24 hours, what was the severity of your overallCOVID-19 related symptoms at their worst?”

1c. Time to sustained recovery, resolution or improvement: comparison intreatment group versus placebo group, respectively of the number of daysfrom randomization to the first day of achieving each item in 1a and 1babove.

2. Physical Functions and Parameters Relevant to COVID-19

2a. Parameter rates: Proportion of subjects in treatment group versusplacebo group, respectively, who on days 4, 7, 10, 14, 21, 28, 42, 60post randomization per thermometer or pulse oximeter readingexperienced:

fever at any point between enrollment and day 2 post randomization andwho were afebrile <38C

oxygenation of SpO2>=96% or >93% in room air when resting

2b. Time to and length of parameters: comparison in treatment groupversus placebo group, respectively of the number of days

from randomization to the first day of achieving sustained (i.e., atleast 2 days) resolution of fever for subjects who presented with feverat any point between enrollment and day 2 post randomization

with temperature <38C or >=38C experienced in total during the first 28days post randomization

from randomization to the first day post randomization of achievingoxygenation of SpO2>=96% in room air when resting for subjects whopresented with SpO2>93% and <96% in room air, when resting, atenrollment

with oxygenation of SpO2>=96% or SpO2>93% in room air, when resting, intotal during the first 28 days post randomization

3. COVID-19 Related Deterioration and Mortality

3a. Deterioration and mortality rates: Proportion of treatment group,versus placebo group, respectively, who per subject reporting or medicalrecords by day 14, day 28 and by day 60 post randomization hadexperienced:

an emergency department visit, other than at study enrollment or studyvisits

hospitalization for COVID-19

hospitalization for COVID-19 requiring oxygen

hospitalization for COVID-19 requiring ICU

hospitalization for COVID-19 requiring ventilation

COVID-19 related death

death

hospitalization or death

3b. Time to and length of deterioration: Comparison of treatment groupversus placebo group, respectively, in the number of days

from randomization until the first day of experiencing hospitalizationfor COVID-19

of hospitalization for COVID-19 experienced in total by day 14, by day28 and by day 60 post randomization

of hospitalization for COVID-19 requiring oxygen experienced in total byday 14, by day 28 and by day 60 post randomization

of hospitalization for COVID-19 requiring ICU experienced in total byday 14, by day 28 and by day 60 post randomization

of hospitalization for COVID-19 requiring ventilation experienced intotal by day 14, by day 28 and by day 60 post randomization

4. WHO 9-Point Ordinal Scale

0. Uninfected or “no clinical or virological evidence of infection”

defined as subject answering “Yes” to “In the past 24 hours, have youreturned to your usual health (before your COVID-19 illness)?”

1. Not hospitalized, no limitations on activities

defined as subject answering “Yes” to “In the past 24 hours, have youreturned to your usual activities (before your COVID-19 illness)?”

2. Not hospitalized, limitation on activities

defined as subject answering “No” to “In the past 24 hours, have youreturned to your usual activities (before your COVID-19 illness)?”

3. Hospitalized, not requiring supplemental oxygen

4. Hospitalized, requiring supplemental oxygen

5. Hospitalized, on non-invasive ventilation or high flow oxygen devices

6. Hospitalized, intubated

7. Hospitalized, advanced life support including invasive mechanicalventilation or ECMO

8. Death

4a) Ordinal scale rates: proportion of treatment group versus placebogroup, respectively who had experienced:

an improvement from scale 2 at randomization to scale 1 or 0 on days 4,7, 10, 14, 21, 28, 42, 60 post randomization,

an improvement from scale 1 at randomization to scale 0 on days 4, 7,10, 14, 21, 28, 42, 60 post randomization

a sustainment from scale 1 at randomization to scale 1 on days 4, 7, 10,14, 21, 28, 42, 60 post randomization

any improvement of the scale (i.e., at least a 1-point decrease) betweenenrollment and days 7, 14, 28, 42, 60 post randomization

any worsening of the scale (i.e., at least a 1-point increase) betweenenrollment and days 7, 14, 28, 42, 60 post randomization

scale 4 or higher by day 28 or day 60 post randomization

scale 6 or higher by day 28 or day 60 post randomization

4b) Ordinal scale averages, highs, and lows: Comparison of treatmentgroup versus placebo group, respectively, at days 14, 28 and 60 postrandomization in the average of daily scale value on that day

overall average of daily scale value experienced since enrollment

in the worst (i.e., highest) daily scale value experienced sinceenrollment

in the best (i.e., lowest) daily scale value experienced sinceenrollment

4c) Time to improvement: Comparison of treatment group versus placebogroup, respectively, in the number of days from enrollment until firstexperiencing a 1-point improvement sustained over at least 2 days by day14, day 28 and by day 60 post randomization

4d) Length of ordinal scale experience: Comparison of treatment groupversus placebo group, respectively, in the total number of days by day14, day 28 and by day 60 post randomization on which subjectsexperienced a given scale value (i.e., 3, 4, 5, 6, 7)

5. Surrogate Markers of COVID-19-Related Inflammation and OrganInvolvement

5a) Analysis and comparison of surrogate markers of COVID-19 relatedinflammation and organ involvement as determined by biochemistry,hematology, and immunology labs and studies, in treatment group versusplacebo group, respectively, for

values on days 1, 7, 14, 21, 28 post randomization

changes from enrollment to days 7, 14, 21, or 28 post randomization

changes between days 7, 14, 21, 28 post randomization

5b) Reference range rates: proportion of treatment group versus placebogroup, respectively, who experience normal/in-range values for a givenmarker at days 7, 14, 21, 28 post randomization

Statistical Considerations Study Populations:

Safety & Tolerability Population: All enrolled subjects who havereceived at least 1 dose of IP (belnacasan or placebo) are studied. Evenif subjects have discontinued study treatment, as long as they maintainconsent, they continue to be in the study and included in analysis,i.e., their safety and tolerability information continues to becollected for at least 60 days.

Treatment Effect Evaluable Populations: All enrolled subjects who havereceived at least 1 dose of IP (belnacasan or placebo) are studied. Evenif subjects have discontinued study treatment, as long as they maintainconsent, they continue to be in the study and included in analysis,i.e., their outcome information continues to be collected for at least60 days.

Analysis Methods:

FDA Data Standard Guidelines are followed. Continuous variables aresummarized using the number of observations, number of observationsabove the limit of quantification (if applicable), mean, standarddeviation (SD) median, and range. Categorical variables are summarizedusing frequency counts and percentages. Comparison of belnacasan andplacebo groups utilize Poisson regression for counts, Cox proportionalhazards regression for time-to-event data and mixed effects models forcontinuous and categorical data obtained over study days.

Primary Safety and Tolerability Analysis:

All safety & tolerability analyses are performed on the SafetyPopulation. The safety data is presented in individual listings andsummary tables. Overall safety is assessed by the number of belnacasandosed subjects experiencing a grade 4 or 5 SAE during the trial. Of 24subjects enrolled in the belnacasan group, 3 or more developing a grade4 or 5 SAE potentially related to belnacasan would be consideredunlikely due to chance. Therefore, belnacasan is determined safe if nomore than 2 subjects develop a grade 4 or 5 SAE in the belnacasan grouppotentially related to belnacasan. If 3 or more subjects in thebelnacasan group develop SAEs potentially related to belnacasan duringthe trial, belnacasan is considered not safe, and the trial stopped orplaced on clinical hold as noted.

Secondary Treatment Effect Endpoint Analysis:

Event counts over 60 days are analyzed by Poisson regression. Time toevents is analyzed by Cox regression. Differences between belnacasan andplacebo with respect to change in continuous endpoints are assessed bymixed effects models for continuous repeated measures data and mixedeffects logistic models for repeated categorical variables. Endpointproportions of belnacasan and placebo groups are assessed by contingencytable analysis (chi-square).

Sample Size Considerations:

As this is a proof-of-concept study, it is likely not be powered todetect belnacasan-placebo differences that are clinically meaningful.For example, the observed difference in endpoint rate ratios would needto be over 60% to achieve 80% power with an alpha error rate of 5% for24 subjects in each group. This study provides an estimate of thebelnacasan effect size to inform a larger trial where a more clinicallymeaningful difference can be detected.

Background and Study Rationale

For more than one year, coronavirus disease 2019 (COVID-19)—an acuterespiratory disease caused by SARS-CoV-2 that also has profoundmulti-systemic inflammatory involvement—has cost countless lives andlivelihoods, especially for vulnerable populations with comorbiditiescharacterized by chronic inflammation. The full spectrum of COVID-19ranges from asymptomatic, to a mild, self-limiting respiratory tractillness, to severe progressive pneumonia, multi-organ failure, and death[1]. Since its first discovery, the virus has continued to spread, evenwith attempted control with a variety of public health measures. Thereare more than 32 million cases in the United States, accounting for aquarter of the worlds documented cases, and over 570,000 deaths [2]. Ingeneral, there is likely an underestimate of total cases since theprevalence of asymptomatic carriers is yet unknown. What is clear, isthat individuals at highest risk are those over 65 years and/or thosewith concomitant chronic conditions most notably hypertension, diabetes,pre-diabetes, obesity, chronic cardiorespiratory disorders and chronicrenal and liver impairment [1, 3-6]. While there are racial disparitiesin the greater number of Black and Hispanic patients experiencing a moresevere course there is no evidence that this is related to anythingother than underlying comorbidities and socioeconomic factors, oftenlimiting their ability to socially distance.

There have been promising scientific breakthroughs for preventing andtreating severe COVID-19, including via vaccines, monoclonal antibodyand steroid treatments. However, there is to-date no targeted treatmentfor SARS-CoV-2-mediated inflammation and subsequent complications inCOVID-19. In general drug treatments aimed at the virus have so far notlived up to their promise. Most notably the nucleotide analog remdesivirwas demonstrated to mitigate the course of the illness when given earlyin the course [7]. Subsequent data however failed to show a benefit.While antibody for treatment has been available the uptake of thistherapy has been limited by parenteral administration. While efficaciousin patients with mild to moderate COVID-19 who are at high risk ofprogressing to severe disease [8], monoclonal antibodies (e.g.,bamlanivimab, casirivimab, and imdevimab) are costly and theiradministration is complicated for outpatients and providers as it isgiven intravenously, in an hour long infusion that must take place in aseparate unit since recipients are infectious. A late 2020 report fromthe Department of Health and Human Services found that only 5-20% ofavailable supply had been used. Currently the most widely accepted andavailable treatment is dexamethasone having shown a clear benefit inseriously affected, hospitalized patients [9].

The most hopeful remedy for the population at large is vaccination andat this time vaccines from three sources (Pfizer/BioNTech, Moderna,Johnson & Johnson) have been authorized for emergency use in the UnitedStates after having demonstrated the ability to prevent disease in up to95% of individuals [10]. However, vaccines may not be sufficient toaddress COVID-19 across the US population for four reasons. First,vaccine hesitancy persists(https://aspe.hhs.gov/pdf-report/vaccine-hesitancy). Second, vaccinationmay only confer immunity for a limited number of months, necessitatingthe need for booster shots(https://www.cnbc.com/2021/04/21/scientist-who-helped-develop-pfizer-biontech-covid-vaccine-agrees-third-shot-is-needed-as-immunity-wanes.html),which may not be taken, or taken on time, by all who need them. Third,immunocompromised people, such as transplant recipients onimmunosuppressive medication, have recently been shown to not developsufficient antibodies through vaccination [11, 12]. Fourth, it remainsto be seen to what extent vaccines are effective against emerging newvariants with immune-escape properties [13-15].

Moreover, given that even mild COVID-19 cases can result in devastating,likely inflammatory-driven, persisting effects(https://www.cdc.gov/coronavirus/2019-ncov/long-term-effects.html),there is clearly an unmet need for a therapeutic option, especially anoral one, that can mitigate harmful uncontrolled inflammation anddysfunctional immune responses during a SARS-CoV-2 infection.

Fortunately, over the past year there has been better understanding ofthe immunologic pathophysiology of COVID-19, which in later stages isdriven primarily by host immune responses to the virus [16]. In thespring of 2020, Applicants' team was the first to publish data linkingthe inflammasome/caspase-1/pyroptosis axis to heightenedpro-inflammatory IL-18 levels, lymphopenia and poor outcome in COVID-19patients [6]. Over the summer of 2020, a Brazilian study in over 100patients expanded Applicants' findings, and also showed direct evidenceof NLRP3 inflammasome activation and caspase-1 mediated pyroptosis inSARS-CoV-2-infected monocytes in vitro, as well as in blood and lungtissue of COVID-19 patients [17, 18]. Inflammasome formation, asevidenced by expression of caspase-1 and NLRP3 in leukocytes andendothelial cells, was also found in the lungs of patients with fatalCOVID-19 pneumonia [19, 20]. Most recently, several studies furthercorroborated and expanded these findings by showing that SARS-CoV-2infects monocytes and directly activates theinflammasome/caspase-1/pyroptosis axis leading to pro-inflammatory celldeath and cytokine release [21, 22]. Importantly, it was furtherdemonstrated that a caspase-1 specific inhibitor was effective inblocking pyroptotic cell death of SARS-CoV-2-infected monocytes andsubsequent release of the pro-inflammatory cytokines IL-1B, IL-6, andTNF-α [21]. In addition, from a mechanistic standpoint, it was recentlyshown that SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3inflammasome and caspase-1 via potassium efflux [23]. Moreover, keyORF3a amino acid residues required for inflammasome activation werefound to be conserved in virus isolates across continents, highlightingthe universality of this inflammatory pathway [23].

Taken together, a growing body of evidence demonstrates that upstreamSARS-CoV-2-mediated inflammasome and caspase-1 activation leading topyroptosis, trigger uncontrolled hyper-inflammation via cytokine release(IL-18, IL-1β, IL-6, and TNF-α) as well as immune dysfunction ofmonocytes and lymphocytes downstream [16, 24-28]. Moreover, activationof the inflammasome/caspase-1/pyroptosis axis could also explainCOVID-19 related organ damage and tissue pathology of the heart, kidney,lungs, liver, pancreas and nervous systems, as it has been shown thatSARS-CoV-2 also appears to cause pyroptosis in a variety of other celltypes, including lung pneumocytes and endothelial cells in these organs[27-32].

Based on this growing body of evidence, this trial proposes that theorally-administered belnacasan, which following hydrolysis on contactwith water is a highly specific caspase-1 inhibitor (also known as IL-1βconverting enzyme) upstream of pyroptosis and pro-inflammatory cytokinerelease, is a much more targeted and potentially safer alternative tocurrent generalized anti-inflammatory COVID-19 treatments such ascorticosteroids, especially for comorbid patients with an alreadychronically-activated inflammasome [6, 25, 27, 28, 33-36] (FIG. 4 ).

Belnacasan (VX-765/RVT-201/MSR-001)

The drug substance has a molecular formula of C24N33CIN406 and hasmolecular weight of 509.0 and comes as a tablet preparation. It ishydrolyzed on contact with water to an active form and is a caspase-1inhibitor (caspase-1 is also known as IL-1β converting enzyme). Blockingthis enzyme with belnacasan (VX-765/RVT-201/MSR-001) has been shown toprevent production of IL-1β, IL-18, and pyroptosis in healthy anddisease states. The drug has been extensively developed originally byVertex Pharma and more recently Roivant Sciences but currently has noFDA or IND approved usage for any indication.

Dosing Rationale

Belnacasan (VX-765/RVT-201/MSR-001) has previously been studied inrodents and large animals including dogs and monkeys to doses of up to2000 mg/kg/day for toxicity studies in dogs. Dosing at these levels andin multi-dosing schedules have not shown any severe and particularlylife-threatening side effects. In human trials, including for conditionssuch as seizure disorders, the drug has been trialed in increments of300 mg/dose on a TID or QID regimen up to 900 mg QID for 13 weeks, innearly 100 patients.

Detailed pharmacokinetic studies from Vertex/Roivant are available forreview. In brief, ex-vivo studies have demonstrated thatLipopolysaccharide stimulated IL-1B production is 50% suppressed by theactive metabolite of belnacasan at a concentration of 0.8 mcg/ml. Thistranslated in to 50% suppression of IL-1B by single oral dose of 400 mgwhile 80% suppression was achieved with doses of 800 and 1600 mg. Therewas gradual but marked inhibition of IL-18 over 14 days in healthycontrols as compared to placebo and 60% inhibition with 900 mg TID aswell as 1200 mg TID and 1800 mg TID during the duration of the study.For this study Applicants have therefore chosen Applicants' primary doseas 900 mg TID in the absence of data on acute viral infection/COVID-19.

The drug has previously been manufactured with appropriate due diligencein tablet form at 300 mg/tablet (Data available from Roivant Sciences)and is manufactured by the same compounding pharmacy (Metrics), in theoriginal format for immediate use in this trial.

For this trial Applicants treat for a maximum of 28 days. Applicants'rationale for this timeframe is that while there is an expectation thatthe illness lasts up to 12-15 days in those who recover withoutcomplication, others endure a longer duration, and some experiencepersistent, new onset or worsening disease related symptoms. A recentstudy from the Centers for Disease Control and Prevention(https://www.cdc.gov/mmwr/volumes/70/wr/mm7017e3.htm?s_cid=mm7017e3_w)found that among 3,171 non-hospitalized adult COVID-19 patients, 69% hadone or more outpatient visits 28-180 days after the diagnosis. Twothirds had a visit for a new primary diagnosis, and approximately onethird had a new specialist visit. Symptoms potentially related toCOVID-19 were common new visit diagnoses. Visits for these symptomsdecreased after 60 days but for some patients continued through 120-180day. Furthermore, some individuals develop sequela that are likelyrelated to post-viral immune dysregulation in the first few monthsalbeit there is little known about this group. Though Applicants areunlikely to learn about the effect of the drug in this latter populationApplicants do need to establish safety for a duration that includes theother groups mentioned above.

Brief Summary of Known Systemic Safety Profile of Belnacasan(VX-765/RVT-201/MSR-001)

Based on files held by Vertex and Roivant Sciences, which containsevidence from animal and human studies, there is no indication thatbelnacasan (VX-765/RVT-201/MSR-001) causes severe or life-threateningside effects. The only preclinical finding on toxicity studies was areduction of the heart rate in dogs at 2000 mg/kg/day, a dose that wasestimated to be 10-25 times higher than the maximum in this study (900mg QID). In humans with epilepsy the drug has been used at dosages of3600 mg/day for up to 13 weeks. Adverse effects reported in humans wereheadache, dizziness, nausea and vomiting, abdominal discomfort and rash.Seizure was reported in more than one patient in one study on epilepsy.

In modern history there is been no greater health threat to life andlivelihood than COVID-19, especially in the United States. Whileprogress is being made to find treatments, there are potential drawbacksto all options. First, even with the prospect of vaccines beingsuccessful, there is a likelihood that patients with co-morbidconditions suffer a protracted illness at least; that it takes time forthe vaccine and any needed boosters to reach critical mass in thepopulation, especially in the face of vaccine hesitancy; and thatvaccines might not always be effective, especially as new virus strandsand variants continue to emerge and early data show thatimmunosuppressed patients may struggle to develop anti-bodies. Second,while the most frequently heralded therapeutic of remdesivir offers thepotential for earlier clearance of the virus, the inflammatory cascaderesults in continued need for inpatient and especially intensive care inhigh-risk patients. Furthermore, as an IV drug, the treatment itselfrequires inpatient treatment and likely needs specific timing to beoptimally effective. Third, the prospect of immunosuppression, forexample with a broad therapy such as dexamethasone or other equivalentcorticosteroid, affects the inflammatory cascade but any use beyond afew days carries risks and therefore disadvantages that Applicantsbelieve come to light with greater use of the drug and scrutiny ofoutcomes going forward. Over the next 12 months it is reasonable toexpect that a large part of the United States population undergoesvaccination with the majority of these individuals being afforded someimmunity for what is yet an undetermined length of time. What is clearhowever is that this form of coronavirus is likely to be around for theforeseeable future and though fewer people are at risk for COVID-19, aneffective treatment is necessary. While antiviral treatment may have aneffect, it is clear from the pathophysiology that clearing virus may notbe enough and such specific therapies to mitigate the individual'sinflammatory response is important to reduce morbidity and mortality.Applicants believe that what is needed is an immunologically targetedmedication that can be taken orally—and without the prospect of majorimmunosuppression in at-risk patients—and that can even be taken forearly treatment in high-risk patients. Based on the data presented hereApplicants believe that belnacasan (VX-765/RVT-201/MSR-001) fulfils thisrole.

Primary Objective

To determine the safety and tolerability of belnacasan(VX-765/RVT-201/MSR-001) administered orally for 28 days in subjectswith mild to moderate COVID-19 as determined by Adverse Events andSerious Adverse Events.

Primary Endpoint

Incidence of Adverse Events and Serious Adverse Events assessed out today 60.

Secondary Objective

To generate data on the treatment effect of belnacasan(VX-765/RVT-201/MSR-001) administered orally for 28 days in subjectswith mild to moderate COVID-19 as determined by its effect size onoutcomes in five areas:

Clinical recovery and resolution of COVID-19 symptoms

Physical functions and parameters relevant to COVID-19

COVID-19 related deterioration and mortality

WHO 9-point ordinal scale for clinical improvement

Surrogate markers of COVID-19-related inflammation and organ involvement

The hypothesized end result of treatment with belnacasan would be toreduce the symptomatic and clinical burden, length of disease course,and disease progression of COVID-19.

Secondary Endpoints

1. Clinical Recovery and Resolution of COVID-19 Symptoms

1a. Sustained recovery and resolution rates of common COVID-19 symptoms:

(i.e., stuffy or runny nose, sore throat, cough, low energy ortiredness, muscle or body ache, headache, chills or shivering, feelinghot or feverish, nausea, vomiting, diarrhea, shortness of breath withexertion; impairment in sense of smell or taste)

Proportion of subjects in treatment group versus placebo group,respectively, who per symptom questionnaire on days 4, 7, 10, 14, 21,28, 42, 60 post randomization have achieved for two consecutive days:

scores not higher than 0 for all symptoms

scores not higher than 1 for all symptoms

scores not higher than 0 for all symptoms other than impairment of tasteor smell

scores not higher than 1 for all symptoms other than impairment of tasteor smell

1b. Sustained improvement of global impression rates: Proportion ofsubjects in treatment group versus placebo group, respectively, who persymptom questionnaire on days 4, 7, 10, 14, 21, 28, 42, 60 postrandomization have answered for two consecutive days

“Yes” to “In the past 24 hours, have you returned to your usual health(before your COVID-19 illness)?”

“Yes” to “In the past 24 hours, have you returned to your usualactivities (before your COVID-19 illness)?”

“None” to “In the past 24 hours, what was the severity of your overallCOVID-19 related symptoms at their worst?”

“Mild” to “In the past 24 hours, what was the severity of your overallCOVID-19 related symptoms at their worst?”

1c. Time to sustained recovery, resolution or improvement: comparison intreatment group versus placebo group, respectively of the number of daysfrom randomization to the first day of achieving each item in 1a and 1babove.

2. Physical Functions and Parameters Relevant to COVID-19

2a. Parameter rates: Proportion of subjects in treatment group versusplacebo group, respectively, who on days 4, 7, 10, 14, 21, 28, 42, 60post randomization per thermometer or pulse oximeter readingexperienced:

fever at any point between enrollment and day 2 post randomization andwho were afebrile <38C

oxygenation of SpO2>=96% or >93% in room air when resting

2b. Time to and length of parameters: comparison in treatment groupversus placebo group, respectively of the number of days

from randomization to the first day of achieving sustained (i.e., atleast 2 days) resolution of fever for subjects who presented with feverat any point between enrollment and day 2 post randomization

with temperature <38C or >=38C experienced in total during the first 28days post randomization

from randomization to the first day post randomization of achievingoxygenation of SpO2>=96% in room air when resting for subjects whopresented with SpO2>93% and <96% in room air, when resting, atenrollment

with oxygenation of SpO2>=96% or SpO2>93% in room air, when resting, intotal during the first 28 days post randomization

3. COVID-19 Related Deterioration and Mortality

3a. Deterioration and mortality rates: Proportion of treatment group,versus placebo group, respectively, who per subject reporting or medicalrecords by day 14, day 28 and by day 60 post randomization hadexperienced:

an emergency department visit, other than at study enrollment or studyvisits

hospitalization for COVID-19

hospitalization for COVID-19 requiring oxygen

hospitalization for COVID-19 requiring ICU

hospitalization for COVID-19 requiring ventilation

COVID-19 related death

death

hospitalization or death

3b. Time to and length of deterioration: Comparison of treatment groupversus placebo group, respectively, in the number of days

from randomization until the first day of experiencing hospitalizationfor COVID-19

of hospitalization for COVID-19 experienced in total by day 14, by day28 and by day 60 post randomization

of hospitalization for COVID-19 requiring oxygen experienced in total byday 14, by day 28 and by day 60 post randomization

of hospitalization for COVID-19 requiring ICU experienced in total byday 14, by day 28 and by day 60 post randomization

of hospitalization for COVID-19 requiring ventilation experienced intotal by day 14, by day 28 and by day 60 post randomization

4. WHO 9-Point Ordinal Scale

0. Uninfected or “no clinical or virological evidence of infection”

defined as subject answering “Yes” to “In the past 24 hours, have youreturned to your usual health (before your COVID-19 illness)?”

1. Not hospitalized, no limitations on activities

defined as subject answering “Yes” to “In the past 24 hours, have youreturned to your usual activities (before your COVID-19 illness)?”

2. Not hospitalized, limitation on activities

defined as subject answering “No” to “In the past 24 hours, have youreturned to your usual activities (before your COVID-19 illness)?”

3. Hospitalized, not requiring supplemental oxygen

4. Hospitalized, requiring supplemental oxygen

5. Hospitalized, on non-invasive ventilation or high flow oxygen devices

6. Hospitalized, intubated

7. Hospitalized, advanced life support including invasive mechanicalventilation or ECMO

8. Death

4a) Ordinal scale rates: proportion of treatment group versus placebogroup, respectively who had experienced:

an improvement from scale 2 at randomization to scale 1 or 0 on days 4,7, 10, 14, 21, 28, 42, 60 post randomization,

an improvement from scale 1 at randomization to scale 0 on days 4, 7,10, 14, 21, 28, 42, 60 post randomization

a sustainment from scale 1 at randomization to scale 1 on days 4, 7, 10,14, 21, 28, 42, 60 post randomization

any improvement of the scale (i.e., at least a 1-point decrease) betweenenrollment and days 7, 14, 28, 42, 60 post randomization

any worsening of the scale (i.e., at least a 1-point increase) betweenenrollment and days 7, 14, 28, 42, 60 post randomization

scale 4 or higher by day 28 or day 60 post randomization

scale 6 or higher by day 28 or day 60 post randomization

4b) Ordinal scale averages, highs, and lows: Comparison of treatmentgroup versus placebo group, respectively, at days 14, 28 and 60 postrandomization in the average of daily scale value on that day

overall average of daily scale value experienced since enrollment

in the worst (i.e., highest) daily scale value experienced sinceenrollment

in the best (i.e., lowest) daily scale value experienced sinceenrollment

4c) Time to improvement: Comparison of treatment group versus placebogroup, respectively, in the number of days from enrollment until firstexperiencing a 1-point improvement sustained over at least 2 days by day14, day 28 and by day 60 post randomization

4d) Length of ordinal scale experience: Comparison of treatment groupversus placebo group, respectively, in the total number of days by day14, day 28 and by day 60 post randomization on which subjectsexperienced a given scale value (i.e., 3, 4, 5, 6, 7)

5. Surrogate markers of COVID-19-related inflammation and organinvolvement

Heart: troponin; Kidney: creatinine, blood urea nitrogen (BUN),electrolytes (calcium, carbon dioxide, chloride, potassium, sodium,magnesium); Pancreas: glucose, HbA1c, lipase; Liver: ALT, AST, ALP,total bilirubin; Muscle weakness: creatinine kinase; Hematology: redblood cell count, hemoglobin, platelet count, prothrombin time (PT),partial thromboplastin time (PTT), d-dimer; Immunology: white blood cellcount, neutrophils, lymphocytes, monocytes, eosinophils, T cells,SARS-CoV-2 viral load in nasopharyngeal sample, SARS-CoV-2 serology inblood; Inflammation: CRP, ferritin, LDH, caspase-1, IL-18, IL-1β, IL-1receptor antagonist, gasdermin D, IL-6, TNF-α, G-CSF

5a) Analysis and comparison of surrogate markers of COVID-19 relatedinflammation and organ involvement as determined by biochemistry,hematology, and immunology labs and studies, in treatment group versusplacebo group, respectively, for

values on days 1, 7, 14, 21, 28 post randomization

changes from enrollment to days 7, 14, 21, or 28 post randomization

changes between days 7, 14, 21, 28 post randomization

5b) Reference range rates: proportion of treatment group versus placebogroup, respectively, who experience normal/in-range values for a givenmarker at days 7, 14, 21, 28 post randomization

The drug in an oral form has been used in nearly one hundred patientswithout a history of major side effects. During the trial, it istherefore anticipated that no more than 2 subjects out of 24 have anadverse event of grade 4 or 5 that is at least possibly related tobelnacasan (VX-765/RVT-201/MSR-001). Should there be 3 or more subjectswith an adverse event grade 4 or 5 that is at least possibly related tobelnacasan (VX-765/RVT-201/MSR-001) in subjects receiving the IP, thestudy is put on an immediate clinical hold. Safety Monitoring processesare identified in Section 7.14.

In addition, individual safety stopping rules include having a non-DLTrespiratory adverse event >grade 3 during study medicationadministration that is at least possibly attributable to belnacasan(VX-765/RVT-201/MSR-001).

Study Description

This is a phase 2 proof of concept randomized, double-blind,placebo-controlled trial with the purpose of assessing the safety,tolerability and treatment effect of the orally administered Caspase-1inhibitor, belnacasan, for the treatment of patients with mild tomoderate COVID-19. 24 subjects are given 900 mg TID of belnacasan and 24subjects are given a placebo TID for 28 days, and assessments of safety,tolerability, and treatment effect are performed for up to 60 days.

At the screening/baseline/day 1 Visit, subjects provide informed consentand be screened for eligibility based on the Inclusion/Exclusioncriteria Subjects also receive the first dose of study drug at thisvisit once study eligibility has been confirmed, and the second dose ofstudy drug is taken approximately 8-12 hours after the first dose. Studydrug is continued three times daily (morning, mid-day, and nighttimedoses) through day 28.

The trial duration is approximately 60 days (8.5 weeks) for all subjectsenrolled. As outlined in the Schedule of Events Table in Section 5.2,over the course of the study duration, all subjects use a diary torecord IP intake as well as symptoms and activity levels with guidanceto do so at the same time each day; assess physical parameters using astudy-provided thermometer and pulse oximeter; be assessed at regularintervals via telemedical and in-person follow-up clinic visits; andgive blood for laboratory tests.

Subjects are instructed to complete the diary at the same time each day,at a time that is consistently convenient for them. The Study Teamconduct in-person visits with the subjects on days 7, 14, 21, and 28 andtelephonic visits with subjects on days 1, 4, 10, 42, and 60. TheTelemetry Care Team conducts telephonic visits daily through day 28.Subjects receive reminders to complete the diary via text message and/oremail. Subjects who prematurely discontinue study drug for any reasonare asked to have an Early Termination (ET) visit the day the last studydrug dose is administered; moreover, they are encouraged to stayenrolled so that further safety and outcome information can be collectedand analyzed.

All data collected by the Study Team and Telemetry Care Team is enteredinto the EDC daily, for this trial OnCore is used. OnCore is a ClinicalTrial Management System in which subject visits can be monitored as wellas Case Report Forms developed. Subjects are asked to hand in theirsubject diary during visits on days 7, 14, 21, and 28.

Background Standard of Care

All eligible subjects receive background standard of care, however thereare certain prohibited medications subjects cannot be given while dosedwith the IP as outlined in Exclusion Criteria. Background standard ofcare are commensurate with disease severity and aligned to standards atthe time of the trial. At present, standard of care includes antiviraltreatment with remdesivir, monoclonal antibodies against SARS-CoV-2,dexamethasone, supplemental oxygen, noninvasive and invasiveventilation, antibiotics, vasopressors and ECMO; but this may evolveover the coming months as new therapies or treatments come online.

Patients recruited to the study are not be asked to forgo backgroundstandard of care in any way. Recruitment is based on inclusion andexclusion criteria and include only individuals evaluated in anemergency room from designated institutions and triaged not to needin-hospital care. These individuals have the potential but not thelikelihood for hospital admission with worsening COVID-19 diseaseprogression. Current outpatient standard of care includes symptomatictreatment and this is not be altered to the point that analgesics andantipyretics are held. Patients whose COVID-19 related illnessdeteriorates to the point that they need ambulatory medical care orhospitalization remain in the study and medical care as determined bymanaging physicians are not subverted in any manner as part of thestudy. Patient data collection as per study diary and study visits aremaintained for the minimum 60 days and study drug is provided andmonitored for compliance unless there is an indication fordiscontinuation of study drug, see section on drug discontinuation.

Study Population

Emergency Department or Critical Care presenting subjects, who are notunder immediate consideration for inpatient admission and with onset ofat least one moderate COVID-19 symptom of less than 7 days, is assessedfor eligibility on the basis of a positive reverse transcriptasepolymerase chain reaction (RT-PCR) assay for SARS-CoV-2 in a respiratorytract sample on presentation to the hospital.

Enrollment of Diverse Study Population

Recognizing that COVID-19 disproportionately impacts racial and ethnicminorities, the study seeks to enroll a diverse patient population. Theenvisioned trial site, MedStar Washington Hospital Center, is thelargest hospital in the District of Columbia. Its primary service areaincludes 11 zip codes, most of which are the zip codes that make upWards 5, 7, and 8. Minorities represent 58% of the population in DCoverall, and 69%, 97%, and 96% of the population in Wards 5, 7, and 8,respectively. Three Metrobuses service the campus and MedStar providesfree, wheelchair-accessible shuttle buses from two Metrorail stations.Moreover, subjects receive financial support for travel to and fromstudy visits.

Number of Subjects

Approximately 48 subjects are recruited at WHC or other MedStar hospitaland/or critical/clinical care sites.

Selection Criteria

Inclusion Criteria:

Subject (or legally authorized representative) provides written informedconsent prior to the initiation of any study procedures.

Subject understands and agrees to comply with planned study procedures,including using the diary.

Subject agrees to the collection of nasopharyngeal swabs and venousblood per protocol.

Subject is male or non-pregnant female adult ≥18 years of age at time ofconsent.

Women with a history of menstruation must agree to use two methods ofcontraception, at least one of which is highly effective, for theduration of the study as well as to undergo additional pregnancy testingduring the study.

Subject has a laboratory confirmed SARS-CoV-2 infection as determined byRT-PCR assay prior to enrollment.

Subject has evidence of either mild or moderate COVID-19 illness of lessthan 7 days from first onset, with minimal baseline symptom severitybased on patient-reported FDA scoring system defined as follows:

Subject presents with at least two common symptoms of COVID-19 from thefollowing list: stuffy or runny nose, sore throat, cough, low energy ortiredness, muscle or body ache, headache, chills or shivering, feelinghot or feverish, nausea, vomiting, diarrhea, shortness of breath withexertion (without supplemental oxygen requirement) with a score of 2 orhigher; impairment in sense of smell or taste with a score of 1 orhigher OR

Subject presents with any (i.e., at least one) symptom of COVID-19 asdefined above AND clinical evidence of moderate COVID-19 as defined byFDA guidance for industry (such as respiratory rate >20 breaths perminute, heart rate >90 beats per minute, with oxygen saturation >93% onroom air at sea level).

Subject presents with high-risk for COVID-19-related inflammationdetermined by at least one comorbidity, including obesity, diabetes,hypertension, stable heart disease, respiratory disease, or non-severefatty liver disease and/or age >60 years.

Subject's overall health condition is deemed as suitable to fully andsafely participate in this trial as determined by the Investigator.

Exclusion Criteria:

Any clinical signs indicative of severe or critical COVID-19 as definedby FDA guidance for Industry at the time, including SpO2<93% and/oroxygen requirement.

Hospitalization for COVID-19, or consideration thereof

ICU level of care and/or non-mechanical/mechanical ventilation and/oroxygen supplementation at time of enrollment.

Pregnant or breast-feeding subjects.

Subjects who cannot swallow tablets.

History of any pre-existing organ impairment, such as:

Severe kidney disease (known or estimated GFR <30 mL/minute) or ondialysis.

Uncontrolled, clinically significant heart diseases such as arrhythmias,angina or heart failure as defined by AHA/ACC Grade C and D.

Chronic respiratory disease requiring supplemental oxygen.

Moderate and severe hepatic impairment as defined by Child-Pugh scoringClass B and Class C

Elevated liver function tests (determined by ALT, AST, GGT, or ALP >2×upper limit of normal, and total Bilirubin >upper limit of normal).

History of malignancy or immunodeficiency within the prior 5 years.

Acute respiratory illness other than COVID-19.

Active bacterial, viral or fungal infection (including HIV, hepatitis B,hepatitis C).

While dosed with the IP, the taking of prohibited concomitant medicationor the ingestion of food that interferes with the IP, including:

Non-COVID19-related anti-viral medication such as lopinavir, ritonavir,ribavirin or interferon-1β.

Systemically administered immunosuppressive and anti-inflammatoryagents, other than background standard of care for COVID-19 at the time.

Drugs and foods that are potent inhibitors or inducers of CYP3A4 and/orP-gp, as listed in FDA “Drug Development and Drug Interactions: Table ofSubstrates, Inhibitors and Inducers”, including herbal medications suchas St. John's Wort within 30 days or 5 half-lives (whichever is longer)prior to the first dose of study drug.

Any other diseases or medical conditions or concomitant medications thatare deemed as not compatible or appropriate for the subject's ability tofully and safely participate in this trial as determined by theInvestigator.

Discontinuation Criteria and Early Termination Procedures

Subjects may withdraw voluntarily from participation in the study at anytime and for any reason. Subjects may also be withdrawn on the basis ofthe Investigator's clinical judgment. This study may be terminated atthe discretion of MedStar Health or of any regulatory agency for reasonsincluding safety and/or treatment effect.

For enrolled subjects Applicants anticipate that the main reasons fordiscontinuation of therapy is either withdrawal of consent forcontinuation in the study or inability to tolerate the study. Otherinstances may include patient death, inability to take the study drugfrom severity of illness or in-hospital complexity of illness.Subjective intolerance of the drug from minor side effects oralternatively cessation of COVID-19 related symptoms well ahead of the28-day schedule may cause patients to want to discontinue the drug. Withregards to drug toxicity itself, Applicants anticipate minorsymptomatology from adverse effects, such as headache, dizziness, nauseaand vomiting, abdominal discomfort and rash (see also 1.4) based onprior human trial experience with the study drug. Should any subjectdosed on the study drug experience seizures, which was reported as anunrelated adverse event in an epilepsy trial, that individual would besubject to discontinuation out of an abundance of caution. Any emergentsymptom that has no clear cause results in a hold on subsequent studydrug dosing until protocol assessment by the Study Investigator. Thereasons for discontinuation of study drug are documented clearly. In allcases study protocol monitoring continues for the entire 60 days of theprotocol as long as the subject stays consented.

When a subject withdraws or is withdrawn before completing the study,the date and reason for withdrawal are to be documented. Subjects whowithdraw or who are withdrawn prematurely are to attend an earlytermination, at which time they complete all assessments as outlined inthe Schedule of Events (Table 1); moreover they are encouraged to stayenrolled so that further safety and outcome information can be collectedand analyzed.

In the event that a subject is withdrawn prematurely due to an adverseevent or serious adverse event, the adverse event or serious adverseevent is followed until it resolves or stabilizes, or until it is judgedby the Investigator to be no longer clinically significant.

Concomitant and Prohibited Medication

At time of enrollment, and at any point during the study, medication orthe ingestion of food that interferes with the IP would precludeparticipation in the study, including:

Non-COVID19-related anti-viral medication such as lopinavir, ritonavir,ribavirin or interferon-1β.

Systemically administered immunosuppressive and anti-inflammatoryagents, other than background standard of care for COVID-19 at the time.

In vitro studies suggest that CYP-mediated metabolism is not a majorroute of clearance for belnacasan (VX-765/RVT-201/MSR-001) orVRT-043198. However, belnacasan (VX-765/RVT-201/MSR-001) was metabolizedby CYP3A4 in a recombinant enzyme system.

Drugs and foods that are potent inhibitors or inducers of CYP3A4 and/orP-gp, as listed in FDA “Drug Development and Drug Interactions: Table ofSubstrates, Inhibitors and Inducers”, including herbal medications suchas St. John's Wort within 30 days or 5 half-lives (whichever is longer)prior to the first dose of study drug

Subjects should also be advised against the consumption of grapefruitjuice since it is a known inhibitor of CYP3A.

Study Drugs

All study medication is managed by the MedStar Health Research Pharmacy(RP). The Pharmacy stores and dispenses medication to subjects.

For all enrolled subjects, the first dose is given orally on day 1 assoon as the subject qualifies and signs the informed consent document.Subjects are closely monitored for at least 30 minutes to assure they donot experience any untoward effect.

For outpatient subjects (and discharged former inpatient subjects),subjects are instructed to take subsequent doses at 0600, 1400, 2200(+/−4 hours). They self-record administration in a subject diary (seeAppendix) to be reviewed by the study team who assesses compliance ateach visit. The study team, under direction of the Investigator,provides any needed medication education, including adequate storagerequirements, with the subject and any relevant family member(s) orcaretaker.

For subjects who become inpatients over the course of the trial, studymedication is supplied to the unit in which subjects are admitted and isdosed by clinical nursing, recorded in the electronic medical record,and overseen by the study team. Doses are given at 0600, 1400, 2200(+/−4 hours). Those who cannot swallow tablets receive crushed tabletssuspended in water and immediately administered through a nasogastrictube, by hospital nursing staff. At discharge and subsequent outpatientvisits, enough medication is dispensed to the subject to assure theyhave adequate supply until their next on-site follow-up visit.

Storage, Dispensing and Reconciliation of Study Drug and Identity ofInvestigational Products

All study medication is stored at room temperature until dispensed.Storage within the RP is locked and secure, accessible only toinvestigational pharmacy staff. Storage condition temperatures arerecorded 24 hours a day, 7 days a week, and 365 days per year and the RPis immediately made aware of any temperature excursions. In such anevent, study medication is not utilized until MedStar Health providesfurther direction. It is the Investigator's responsibility to ensurethat accurate records of study medication dispensation andadministration are maintained. The RP supports the Investigator inmedication accountability and dispensation tracking, they have a clearprocess for return and destruction of investigational medications.

TABLE Identification of Investigation Product Product Name Belnacasan(VX-765/RVT-201/MSR-001) Dosage form Tablet containing 300 mg of APIRoute/dosage Oral Dosing Instructions Three tablets, three times per day(0600, 1400, 2200 +/− 4 hours) Product Name Placebo Dosage form Tabletcontaining 0 mg of API Route/dosage Oral Dosing Instructions Threetablets, three times per day (0600, 1400, 2200 +/− 4 hours)

Observations and Measurements

Subject informed consent must be obtained prior to conducting anystudy-specific procedures. The informed consent can be signed by thesubjects' legally authorized representative (LAR) if necessary. TheInvestigator assures that each subject/LAR is adequately consented tothe requirements of participation including the potential risks andbenefits and voluntary nature of the trial that he or she is free todiscontinue participating in the study at any time. The subject/LAR isgiven the opportunity to ask questions and allowed adequate time toconsider the information provided. All assessments and procedures arecompleted according to the Schedule of Events. A custom subject diaryhas been developed for this study. The diary is tested for basiccomprehensibility before finalizing and using with subjects.

Instructions to Subjects

At enrollment, the Study Team explains the subject diary (see Appendix)and ensure they understand what to enter in each field. They are told ofthe importance of collecting the information in the diary for scientificpurposes, and that even if they discontinue intake of the study drug,they should continue to participate in the study, continue to fill inthe diary, and continue to attend planned study visits.

For outpatient subjects (and discharged former inpatient subjects),subjects are instructed to take 3 tablets of IP at 0600, 1400, 2200(+/−4 hours) for up to 28 days. They self-record administration of theIP in the subject diary to be reviewed by the study team who assessescompliance at each visit. The study team, under direction of theInvestigator, provide any needed medication education, includingadequate storage requirements, with the subject and any relevant familymember(s) or caretaker.

For subjects who become inpatients over the course of the trial, studymedication are supplied to the unit in which subjects are admitted andare dosed by clinical nursing, recorded in the electronic medicalrecord, and overseen by the study team. Doses are given at 0600, 1400,2200 (+/−4 hours). Those who cannot swallow tablets receive crushedtablets suspended in water and immediately administered through anasogastric tube, by hospital nursing staff. As study medication isadministered by hospital staff, and there are no specific instructionsto subjects. At discharge and subsequent outpatient visits, enoughmedication is dispensed to the subject to assure they have adequatesupply until their next on-site follow-up visit.

Women with a history of menstruation must agree to use effective methodof contraception and protections that align with International Councilon Harmonization M3 R2 guidelines for highly effective methods ofcontraception for the duration of the study (i.e., two forms ofcontraception, whereby one result in a less than 1 percent per yearfailure rate when used consistently and correctly), as well as toundergo additional pregnancy testing during the study on day 14. Detailsof all pregnancies in female participants are collected for 60 daysafter randomization.

Warnings and Precautions

Subjects are watched carefully for signs of previously documentedsymptoms in human patients that have received belnacasan(VX-765/RVT-201/MSR-001), as well as symptoms to indicate a drugreaction or new symptoms that could be temporally attributable to thedrug. The most frequent adverse effects in humans have been mild andmainly include headache, nausea, lethargy and dizziness. No serious sideeffects have been described in human and animal studies.

NCI CTCAE Definitions of Dose Limiting Adverse Events

The National Cancer Institute (NCI), Common Terminology Criteria forAdverse Events (CTCAE) are used for monitoring adverse events throughoutstudy including those related to dose limitation. As noted above,adverse events with belnacasan (VX-765/RVT-201/MSR-001) have been mildand of no consequence (grade 1 and 2 by CTCAE criteria). There is nodose escalation in this trial that could result in new symptoms thoughit is possible that mild symptoms may become more severe.

Pre-Existing Medical Conditions

All subjects enrolled in the study have SARS-CoV-2 infection and atleast two moderate COVID-19 symptoms or one moderate symptom andclinical evidence of moderate COVID-19. Subjects present with high-riskfor COVID-19-related inflammation, due to at least one comorbidity,including obesity, diabetes, hypertension, stable heart disease,respiratory disease, or non-severe fatty liver disease and/or age >60years.

Acute Conditions Brought on by COVID-19.

The development of acute kidney and/or hepatic injury has been reportedas a possible complication during the disease progression of COVID-19.Acute kidney and/or hepatic injury are addressed in the following mannerdepending on whether at, (1) enrollment, (2) during the study periodwhen drug is being administered in an ambulatory setting, (3) or duringadmission to hospital for COVID-19 related deterioration or otherillness.

Potential participants in this study undergoes initial evaluation in theemergency room that includes background laboratory testing. If thetesting indicates renal failure, GFR <30 or features of acute renalfailure, most notably anuria, edema on examination in a previouslyhealthy individual, the potential participant are not be enrolled as perthe inclusion criteria or otherwise clarified with the nephrologyconsultant managing the patient. Similarly, potential participants shownto have evidence of elevated liver function tests (determined by ALT,AST, GGT, or ALP >2× upper limit of normal, and total Bilirubin >upperlimit of normal) is not be enrolled in the study.

Laboratory testing throughout the study period may, though unlikely,show an acute change in renal or liver testing to indicate acuteorgan-specific disease or involvement as a possible complication ofCOVID-19. In such a circumstance, subjects are directed to seek carefrom the respective hospital/specialist. In the case of acute liverinjury with functional impairment, the IP is discontinued immediatelyafter blood has been taken for future assessment of drug levels. In thecase of subjects with elevated liver function tests suggestive of acuteliver injury without functional impairment, IP is held to discern in thefirst instance the possibility of an adverse event and restarted inconsultation with the managing hepatologist if it is determined to beCOVID-19 related hepatopathy. In the case of subjects with acute renalinjury, IP is held to discern in the first instance the possibility ofan adverse event and restarted in consultation with the managingnephrologist if it is determined to be COVID-19 related nephropathy.DSMB guidelines for unblinding is followed in these cases as relevant.

Acute liver and kidney injury may be seen in the face of hospitaladmission in patients with worsening COVID-19, in which case thepatients are managed similarly to number 2 above (also seediscontinuation).

Treatment Emergent Adverse Events

A treatment-emergent adverse event (TEAE) is defined as any event notpresent prior to the initiation of the treatments or any event alreadypresent that worsens in either intensity or frequency following thestart of IP administration.

Adverse events are captured following the first dose of IP and could berelated or unrelated to the study drug. A TEAE is defined as any AE thatoccurs after the subject takes the first dose of IP.

Separate summaries for adverse events that occur during treatment(summary of treatment emergent adverse events) are provided.

Laboratory Abnormalities

Clinical labs are performed locally at WHC or other MedStar clinicalsites. Labs to be drawn during the study include serum chemistries, ahematology panel and an immune panel. A serum pregnancy test must beperformed, and the result must be negative prior to the entry of womenof child-bearing potential.

Clinical laboratory reports must be reviewed by a physician forout-of-range values within 12 hours of receipt. Out-of-range values areevaluated using the following notations:

NCS: Not clinically significant

LE: Laboratory Error

PT: Subject abnormal; relates to the subject's usual state of health

CS: Clinically Significant. This value cannot be explained by any of theother indicators.

By definition a lab value flagged as “CS” indicates an adverse event andare captured on the CRF. A laboratory test flagged “CS” should berepeated as soon as possible, then the Investigator should use his orher judgment as to whether the abnormal finding is sufficient reason toimmediately withdraw the subject from the study.

If a laboratory value is considered to be serious and life-threateningand at least possibly related to the study drug, the subject should beimmediately discontinued from the study and appropriate therapy started.

Adverse Event Assessment and Recording

All adverse events, exacerbations of concomitant illnesses, or eventsknown to be related to underlying disease processes or concomitantmedications are to be recorded on the CRF throughout the study. If apre-existing condition worsens during the study, the date on which theexacerbation began should be recorded. Onset dates for studytreatment-related adverse events must be on or after the date of initialstudy treatment use.

Adverse event recording includes the date of onset, severity, duration,whether or not the study medication was discontinued because of theevent, the treatment given, and the outcome. The Investigator must alsoassess whether the event was related to the study medication, concurrentdrug therapy, underlying disease, a combination of these factors, or ifit is unknown. Subjects experiencing an adverse event should becarefully followed to determine outcome.

The Investigator use the NCI-CTCAE for adverse event monitoring, version5.0 is applied in all instances. CTCAE version 4.03 may also be usedespecially when assessing laboratory tests given the complexity inapplying version 5.0 in all instances, seewww.lexjansen.com/phuse-us/2020/dh/DH16.pdf

Definitions to grade the severity of the event:

Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnosticobservations only; intervention not indicated.

Grade 2: Moderate; minimal, local, or non-invasive interventionindicated; limiting age-appropriate ADL.

Grade 3: Severe or medically significant but not immediatelylife-threatening; hospitalization or prolongation of hospitalizationindicated; disabling; limiting self-care ADL.

Grade 4: Life-threatening consequences; urgent intervention indicated.

Grade 5: Death.

The relationship or association of the study medication in causing orcontributing to the adverse event is characterized as not related,remote, possible, probable, or definite as defined below:

Not related: Evidence indicates no plausible direct relationship to thestudy medication

Remote: Suggests other conditions are reasonably likely to account forthe event including concurrent illness, progression or expression of thedisease state, or reaction to concurrent medication

Possible: Suggests that the association of the event with the studymedication is unknown; however, the adverse event is not reasonablysupported by other conditions

Probable: Suggests that a reasonable temporal sequence of the event withmedication administration exists and based upon the Investigator'sclinical experience, the association of the event with study medicationseems likely

Definite: Suggests that based upon the Investigator's experience, theassociation of the event with the study medication seems very certain.

Planned procedures such as surgery should not be recorded as adverseevents. However, the medical condition for which the procedure wasperformed should be reported if it meets the definition of adverseevent.

Reporting Requirements

Any adverse event, defined below, that begins any time between the startof the first dose and within 28 days after the end of the last dose areto be recorded on the appropriate CRF and in detail in the sourcedocumentation (preferably the electronic medical record directly).

Adverse Event: Any untoward medical occurrence in a subject administereda pharmaceutical product, which does not necessarily have to have acausal relationship with this treatment. An adverse event can be anyunfavorable sign (including an abnormal laboratory finding), symptom, ordisease temporally associated with the use of the investigational drug,whether or not considered related to the investigational drug.

Serious Adverse Event: An untoward event or reaction that at any dose:

results in death

is life-threatening

prolongs existing hospitalization

results in permanent or significant disability or incapacity

requires intervention to prevent permanent impairment/damage

Life-threatening: An event which a subject was at risk of death at thetime of event. There is a distinction between the severity and theseriousness of an adverse event. Severity is a measurement of intensity,thus a severe reaction is not necessarily a serious adverse event. Forexample, a headache may be severe in intensity, but would not be seriousunless it met one of the criteria for serious adverse events listedpreviously.

Serious Adverse Events

Adverse events (AEs) and serious adverse events (SAEs) is collected fromthe time the first dose of IP is administered until 28 days after thelast dose of IP. Medical occurrences that began prior to the start ofstudy treatment, but after obtaining informed consent is captured asMedical History/Current Medical Conditions on the CRF. The Investigatoror site staff is responsible for the identification and documentation ofevents meeting the criteria and definition of an adverse event orserious adverse event, as provided in the study protocol. SAEs assessedas related to study participation (e.g., dosing, protocol mandatedprocedures, invasive tests, or change in existing therapy) or related toa concomitant medication is captured once the subject has taken thefirst dose of study medication.

In the event of an AE or SAE, it is the responsibility of theInvestigator to review all documentation (e.g., hospital progress notes,laboratory, and diagnostics reports) relative to the event and attemptto establish a diagnosis of the event based on signs, symptoms, andother clinical information. Once the Investigator becomes aware that anSAE has occurred, they are′ to report the information to MedStar Healthwithin 24 hours and provide an assessment of causality.

Notification of Serious Adverse Events

The Sponsor reports all serious adverse events to regulatory agencies asrequired. In addition to the serious adverse events describedpreviously, other events that in the Investigator's opinion suggest asignificant hazard, contraindication, or precaution should be consideredserious. This includes, but is not limited to, blood dyscrasias,endocrine disturbances, hemorrhage from any site, or severe skindisorder. Additional examples are intensive treatment for allergicbronchospasm, blood dyscrasias or convulsions.

Subjects who experience an SAE must be given appropriate examinationsand treatment. The Investigator must provide written information to theSponsor as soon as possible.

Reporting a Serious Adverse Event

All appropriate SAEs are reported immediately to the FDA per reportingcriteria for an IND Safety Report. The event(s) are reported locally tothe IRB of record when the event meeting reporting criteria per MedStarHealth Research Institute IRB policy.

Departure from Protocol for Emergency or Adverse Event

In medical emergencies, the Investigator should use medical judgment andremove the subject from immediate hazard. As soon as possible afterremoving the subject from hazard, the Investigator must contact MedStarHealth by telephone to permit a decision as to whether the subject maycontinue in the study. The IRB should also be notified as to the type ofemergency and the course of action. The CRF for the subject must capturethe departure from the protocol and state the reason.

Safety Monitoring

A Data Safety and Monitoring Board (DSMB) is established to monitor thesafety of the subjects during the study. The DSMB includes members whoare independent of this study and its Sponsor Team, and with relevantclinical expertise, including a good understanding of the safety ofmedications for respiratory illnesses. These members may include one ormore of the following—a statistician, an infectious disease specialistand/or a pulmonologist. The methodology and the operating procedures forthe safety reviews are developed by the infectious disease specialistand/or pulmonologist in collaboration with the Investigator and aredocumented in the DSMB Charter. They review all SAEs and determinewhether the study can proceed or whether protocol modifications arerequired.

Stopping Rules

During the study it is anticipated that no more than 2 subjects out of24 have an adverse event of grade 4 or 5 that is at least possiblyrelated to belnacasan (VX-765/RVT-201/MSR-001). Should there be morethan 3 subjects with an adverse event grade 4 or 5 that is at leastpossibly related to the IP, the study is put on an immediate clinicalhold.

In addition, individual safety subject study stopping rules includehaving a non-DLT respiratory adverse event >grade 3 within 24 hours oftaking study medication that is at least possibly attributablebelnacasan (VX-765/RVT-201/MSR-001).

Follow-Up and Final Reports

The Investigator shall provide MedStar Health with an accurate finalreport within 1 month after completion, termination or discontinuationof the study. The final report may not precede completion of monitoringrelevant CRFs.

Regulatory Aspects

Neither the Investigator nor MedStar Health shall modify this protocolwithout first obtaining concurrence of the other in writing. Allmodifications must be submitted to the IRB with approval prior toimplementation. Protocol modifications which impact subject safety orthe validity of the study must be approved by the IRB and submitted tothe FDA before implementation. In the case of a medical emergency toincrease safety of subjects, a change may occur immediately, and the IRBand FDA are notified as soon as possible.

Populations for Analyses

The following populations are considered for statistical analyses.

Safety & Tolerability Population: All enrolled subjects who havereceived at least 1 dose of IP (belnacasan or placebo) are studied. Evenif subjects have discontinued study treatment, as long as they maintainconsent, they continue to be in the study and included in analysis,i.e., their safety and tolerability information continue to be collectedfor at least 60 days.

Treatment Effect Evaluable Populations: All enrolled subjects who havereceived at least 1 dose of IP (belnacasan or placebo) are studied. Evenif subjects have discontinued study treatment, as long as they maintainconsent, they continue to be in the study and included in analysis,i.e., their outcome information continue to be collected for at least 60days.

Analysis Methods

General Methods

FDA Data Standard Guidelines are followed. Continuous variables aresummarized using the number of observations, number of observationsabove the limit of quantification (if applicable), mean, standarddeviation (SD) median, and range. Categorical variables are summarizedusing frequency counts and percentages. Comparison of belnacasan(VX-765/RVT-201/MSR-001) and placebo groups utilize Poisson regressionfor counts, Cox proportional hazards regression for time-to-event dataand mixed effects models for continuous and categorical data obtainedover study days.

Analysis of Subject Disposition, History, and Baseline Characteristics

Subject disposition, including analysis population allocation, subjectsenrolled, completed each period, discontinued, and primary reason fordiscontinuation, are summarized using frequency and percentage. Protocoldeviations are summarized using frequency and percentage. Medicalhistory data and prior and concomitant medications are summarized usingfrequency and percentage. Subjects' age, height, weight, and baselinedisease characteristics are summarized using descriptive statistics.Gender, race, and other categorical variables are provided usingfrequency and percentage.

Safety & Tolerability Analyses

All safety & tolerability analyses are performed on the SafetyPopulation. The safety dataise presented in individual listings andsummary tables.

Overall safety is assessed by the number of belnacasanVX-765/RVT-201/MSR-001 dosed subjects experiencing a grade 4 or 5 SAEduring the trial. Of 24 subjects enrolled in the belnacasan group, 3 ormore developing a grade 4 or 5 SAE potentially related to belnacasanwould be considered unlikely due to chance.

Therefore, belnacasan is determined safe if no more than 2 subjectsdevelop a grade 4 or 5 SAE in the belnacasan group potentially relatedto belnacasan. If 3 or more subjects in the belnacasan group developSAEs potentially related to belnacasan during the trial, belnacasan isconsidered not safe, and the trial stopped or placed on clinical hold asnoted.

Primary Endpoint Analysis

Primary endpoints of adverse and serious adverse events (AE/SAE) isassessed by Poisson regression for count data. Events are counted out to60 days from randomization to belnacasan or placebo groups. Because somesubjects may be followed for less than 60 days, subject follow-up timeare included in the Poisson regression as the exposure time variable. Ifcounts are not Poisson-distributed, either over- or under-dispersed,negative binomial regression analyze AE/SAE events with appropriateadjustment for over/under dispersion. Comparison of belnacasan vs.placebo is expressed as the incidence rate ratio (IRR).

Adverse Events

AEs are coded according to the latest version of the Medical Dictionaryfor Regulatory Activities (MedDRA). The intensity/severity of AEs isgraded according to NCI CTCAE.

TEAEs, AEs leading to study treatment discontinuation, AEs leading todose interruption, AEs related to study medication, SAEs, and AEsleading to death are summarized by system organ class, preferred term,and study period. A summary of AEs that are CTCAE Grade 3 or higher, aswell as the most frequent preferred terms, are provided.

If a subject experiences the same adverse events multiple times within aperiod, then the event is counted only once within the period and bygreatest severity.

Descriptive statistics are used to summarize the safety data.

Clinical Laboratory Values

All laboratory test results are summarized by period together with thechange from baseline. The frequency distribution for low/normal/high ornormal/abnormal are summarized as well. The denominators for calculatingthe percentages are based on the number of subjects with non-missingvalues in the Safety Population.

Vital Signs

Vital sign results are summarized by period, together with the changefrom baseline.

Physical Examination

Summaries of physical examinations present frequency distribution ofabnormal findings by body system and period. The denominators forcalculating the percentages are based on the number of subjectsevaluated for a particular body system of each dose level in the SafetyPopulation.

Electrocardiogram (EKG)

EKG findings are classified as normal vs abnormal. The number andpercentage of each category is summarized using frequency table for eachperiod. The denominators for calculating the percentages are based onthe number of subjects with non-missing values in each period.

Treatment Effect Analyses

Secondary Endpoint Analyses

Event counts over 60 days are analyzed by Poisson regression asdescribed above in 8.4. Time to events are analyzed by Cox regression.Differences between belnacasan and placebo with respect to change incontinuous endpoints are assessed by mixed effects models for continuousrepeated measures data and mixed effects logistic models for repeatedcategorical variables. Endpoint proportions of belnacasan and placebogroups are assessed by contingency table analysis (chi-square).

Handling of Missing Data

Although every effort is made to obtain complete data, missing valueslikely occur when data are collected longitudinally. Patterns ofmissingness over time as well missingness with respect to demographicand clinical variables are assessed. For Poisson and Cox regressionanalyses, all data can be used up to subject dropout time.

Sample Size Considerations

A total of 48 subjects are randomized, 24 to belnacasan(VX-765/RVT-201/MSR-001) 900 mg TID and 24 to placebo. As this is aproof-of-concept study, it is likely not powered to detectbelnacasan—placebo differences that are clinically meaningful. Forexample, the observed difference in primary endpoint rate ratios wouldneed to be over 60% to achieve 80% power with an alpha error rate of 5%for 24 subjects in each group. This study provides an estimate of thebelnacasan effect size to inform a larger trial where a more clinicallymeaningful difference can be detected.

This study has an estimated maximum duration of up to 8.5 weeks for eachsubject. The study duration from first subject enrolled to last subjectenrolled is expected to be 3-6 months.

Subject Information and Informed Consent

The Investigator ensures that the subject/LAR fully understands studyparticipation requirements including possible risks and benefits.Subjects must also be notified that they are free to withdraw from thestudy at any time. During the informed consent process, the subjectshould be given the opportunity to ask questions and allowed time toconsider the information provided. The subjects' informed consent mustbe obtained before conducting any study-specific procedures.

Study Monitoring

During the study, a dedicated Clinical Research Associate (CRA) haveregular contacts with the investigational site, for the following:

Provide information and support to the Investigator,

Confirm that facilities remain acceptable,

Confirm that the investigational team is adhering to the protocol, thatdata is being accurately recorded in the eCRFs, and that investigationalproduct accountability checks are being performed,

Perform source data verification. This includes a comparison of the datain the eCRFs with the subjects' medical records, and other recordsrelevant to the study. This requires access to all original records foreach subject (e.g., clinic charts) as described in the study monitoringplan.

Record and report any protocol deviations not previously sent to MedStarHealth.

Confirm adverse events and serious adverse events have been properlydocumented on eCRFs and confirm any serious adverse events have beenforwarded to MedStar Health, and those serious adverse events that metcriteria for reporting have been reported to the IRB.

The CRA is available over the course of the study if the Investigator orother staff needs information or advice. Remote visits are conducteduntil it is possible to perform on-site visits.

Audits and Inspections

Authorized representatives of the FDA, or the Institutional Review Board(IRB) may visit the site to perform audits or inspections, includingsource data verification. The purpose of any inspection is tosystematically and independently examine all study-related activitiesand documents to determine whether these activities were conducted, anddata were recorded, analyzed, and accurately reported according to theprotocol, Good Clinical Practice (GCP) guidelines of the InternationalConference on Harmonization (ICH), and FDA regulations. TheInvestigator/Sponsor should contact the site immediately if contacted bya regulatory agency about an inspection.

Ethics Committee Review

The final study protocol, including the final version of the InformedConsent Form, must be approved by the MedStar Health Research InstituteIRB.

The Sponsor is responsible for submitting any modifications to theProtocol to the IRB and obtaining approval in advance of implementingthese modifications. In addition, the IRB must approve all advertisingused to recruit subjects for the study. The study must also be renewedannually with the IRB as long as study conduct is occurring.

The Investigator/Sponsor are also responsible for providing the IRB withreports of any reportable new information (RNI) including any seriousadverse drug reactions from any other study conducted with theinvestigational product.

Standards

The study is performed in accordance with ethical principles that havetheir origin in the Declaration of Helsinki and are consistent withICH/GCP and applicable regulatory requirements.

Confidentiality

Any research information obtained specific to subjects enrolled intothis study are kept confidential. A subject are not identified by name,only by a unique study number. The subject's name or any identifyinginformation does not appear in any reports published as a result of thisstudy.

However, information obtained from individual subjects' participation inthe study may be disclosed with his/her consent to the healthcareproviders for the purpose of obtaining appropriate medical care. Thesubject's medical records/charts, tests with his/her name on them may bemade available to the appropriate contract research organization (CRO),MedStar Health, IRB, and the FDA. This is for the purpose of verifyinginformation obtained for this study.

A subject's name is not to be given to anyone except the researchersconducting the study, who have pledged an oath of confidentiality. Allidentifying information is kept behind locked doors, under thesupervision of the Investigator and is not to be transferred outside ofWHC/MedStar as applicable.

A subject may take away his/her permission to collect, use and shareinformation about him/her at any time. If this situation occurs, thesubject is not able to remain in the study. No new information thatidentifies the subject is gathered after that date. However, the dataabout the subject that has already been collected may still be used andgiven to others as described above in order to preserve the scientificintegrity and quality of the study.

Protocol Adherence

The records of study treatment delivered to subjects and IP inventory ismaintained at WHC/MedStar; the administration to each subject; andstorage or disposal of any unused IP. These records should includedates, quantities, batch/serial numbers, expiration dates, IDStemperature log, and unique code numbers assigned to the product andstudy subjects.

The Investigator maintains records that document adequately that thesubjects were provided with the correct study drug and maintains IPaccountability and tracking records.

Amendments to the Protocol

Modifications to the protocol are only possible by approved protocolamendments authorized by the Sponsor, approved by the IRB, and deemed asnot-objectionable by the FDA. The Investigator must not implement anydeviations from, or changes to the protocol, except where it isnecessary to eliminate an immediate hazard to the study subject.

Protocol Deviations

The protocol must be conducted compliantly, significant deviations fromthe protocol is reported to the FDA and/or IRB per policy.

Study Termination

This study may be prematurely terminated, if in the opinion of theInvestigator or MedStar Health there is sufficient reasonable cause.Circumstances that may warrant termination include, but are not limitedto:

Determination of unexpected, significant, or unacceptable risk tosubjects.

Failure to enroll subjects at an acceptable rate.

Insufficient adherence to protocol requirements.

Insufficient complete and/or evaluable data.

Plans to modify, suspend, or discontinue the development of belnacasan(VX-765/RVT-201).

Inspection of Records

MedStar Health is allowed to audit data for the purpose of monitoringany aspect of the study they deem necessary, pursuant to localrestrictions on monitoring due to health concerns. The Investigatoragrees to allow the IRB and regulatory authorities to inspect the drugstorage area, study drug stocks, drug accountability records, subjectcharts and study source documents, and other records relative to studyconduct. Remote access can be granted in the event the pandemic preventson-site monitoring.

Data Management

All data relating to study procedures is entered onto the eCRF (OnCoreplatform). CRF completion guidelines are developed to assure therequired data is appropriately captured. eCRFs are completed for eachsubject. It is the Investigator's responsibility to ensure the accuracy,completeness, and timeliness of the data entered in each subject's eCRF.Source documentation supporting the eCRF data should indicate thesubject's participation in the study and document the dates and detailsof study procedures, adverse events, and subject status.

The Investigator, or designee, should complete the eCRF as soon aspossible after data is collected, preferably on the same day that asubject is seen for an examination, treatment, or any other studyprocedure. Any outstanding entries must be entered immediately after thefinal examination. An explanation should be given for all late ormissing data.

Liability and Insurance

MedStar Health has subscribed to an insurance policy covering, in itsterms and conditions, its legal liability for certain injuries toparticipating persons arising out of this research performed strictly inaccordance with the scientific protocol as well as with applicable lawand professional standards.

Retention of Records

Investigators shall retain study records and source documents for aperiod of 2 years following the date a marketing application is approvedfor the drug for the indication for which it is being investigated; or,if no application is to be filed or if the application is not approvedfor such indication, until 2 years after the investigation isdiscontinued and FDA is notified and/or the period required by local,state, and federal laws, regulations and FDA Guidance.

The Investigator agrees to comply with all applicable federal, state,and local laws and regulations relating to the privacy of protectedhealth information, including, but not limited to, the Standards forIndividually Identifiable Health Information, 45 CFR, Parts 160 and 164(the Health Insurance Portability Accountability Act of 1996 [HIPAA]Privacy Regulation). The Investigator shall ensure that study subjectsauthorize the use and disclosure of protected health information inaccordance with HIPAA Privacy Regulation.

Data Quality Assurance

WHC/MedStar and all relevant subject study and medical records may besubject to a quality assurance audit during the course of the study. Inaddition, inspections may be conducted by the FDA at their discretion.

Both the use of data and the publication policy are detailed within theclinical study agreement. Intellectual property rights (and relatedmatters) generated by the Investigator and others performing theclinical study are subject to the terms of a clinical study agreementthat is agreed between the Institution and MedStar Health or theirdesignee. With respect to such rights, MedStar Health or its designeesolely owns all rights and interests in any materials, data, andintellectual property rights developed by Investigators and othersperforming the clinical study described in this protocol, subject to theterms of any such agreement. In order to facilitate such ownership,Investigators are required to assign all such inventions directly toMedStar Health or its designee, as are set forth in the clinical studyagreement.

Appendix

Labs and Tests Hematology Serum Chemistry Complete Blood Count: AlbuminWhite Blood Cell (WBC) Count Alanine Aminotransferase (ALT) Red BloodCell (RBC) Count Alkaline Phosphatase (ALP) Hemoglobin (Hb) AspartateAminotransferase (AST) Hematocrit (Hct) Blood Urea Nitrogen (BUN) MeanCorpuscular Volume (MCV) Calcium Red Blood Cell Distribution WidthCarbon Dioxide (RDW) Chloride Platelet Count Creatinine Differential -absolute and Glucose percent of: Lipase Neutrophils MagnesiumLymphocytes Potassium Monocytes Sodium Eosinophils Total BilirubinBasophils Total Protein Coagulation Tests: Lactate Dehydrogenase (LDH)Partial Thromboplastin Time (PTT) Creatine Kinase, Total ProthrombinTime (PT) CRP Immune Cell Counts - absolute and Ferritin percent of:D-Dimer CD3+ T cells HbA1c Serum and Plasma Levels of Screening TestsCytokines and Inflammasome HIV Markers: Hepatitis B IL-18 Hepatitis CIL-1β Pregnancy (serum pregnancy test for IL-1 receptor antagonist womenof child-bearing potential IL-6 per MedStar definition) Gasdermin DG-CSF TNF-α Caspase-1

FIGS. 5A-5C provide a Subject Diary.

A randomization table such as the one below are used to allocatesubjects to belnacasan or placebo group (N=24 each). Assignment of A orB to treatment or placebo blinded to subjects and investigators.

Sequential Group Sequential Group subject assignment subject assignment1 A 25 B 2 B 26 A 3 B 27 A 4 A 28 B 5 A 29 A 6 B 30 B 7 A 31 A 8 B 32 B9 A 33 A 10 A 34 A 11 B 35 B 12 B 36 B 13 B 37 B 14 A 38 B 15 A 39 A 16B 40 A 17 B 41 B 18 A 42 A 19 B 43 B 20 A 44 A 21 B 45 A 22 B 46 B 23 A47 B 24 A 48 A

The invention is further described by the following numbered paragraphs:

1. A method for treating a patient infected with SARS-CoV-2 or a variantthereof and having pyroptotic activity comprising (a) determining if thepatient is infected with SARS-CoV-2 or a variant thereof, (b) optionallydetermining if the patient has pyroptotic activity or a comorbidityassociated with upregulation of an inflammasome/caspase/pyroptosis axis,and (c) administering an effective amount of a compound having theformula

to the patient infected with SARS-CoV-2 or a variant thereof.

2. The method of numbered paragraph 1, wherein step (b) comprisesscreening a marker indicative of pyroptotic activity, wherein the markerhas increased activity if pyroptotic activity is present.

3. The method of numbered paragraph 2, wherein the marker is NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3), IL-1 receptor antagonist(IL-1RA), interleukin-1β (IL-1β), interleukin-18 (IL-18), gasdermin D(GSDMD), caspase 1, lymphopenia, a high neutrophil count, elevated LDH,ferritin or CRP levels.

4. The method of any one of numbered paragraphs 1-3, wherein theeffective amount in step (c) is about 10 mg/kg to about 100 mg/kg.

5. The method of numbered paragraph 4, wherein the effective amount is300 mg to about 900 mg per dose.

6. The method of numbered paragraph 5, wherein the effective dose isabout 600 mg to about 900 mg per dose.

7. The method of any one of numbered paragraphs 4-6, wherein theeffective dose is about 300 mg to about 3600 mg per day.

8. The method of any one of numbered paragraphs 5-7, wherein the 300 mgto about 900 mg is administered three or four times a day.

9. The method of numbered paragraph 8, wherein the administering isabout every six to about eight hours.

10. The method of any one of numbered paragraphs 1-9, wherein theadministering in step (c) is oral.

11. The method of numbered paragraph 10, wherein the oral administeringis a tablet.

12. The method of numbered paragraph 10, wherein the tablet comprises a300 mg dose.

13. The method of numbered paragraph 12, wherein the administeringcomprises one tablet, two tablets or three tablets.

14. The method of any one of numbered paragraphs 1-13, wherein theadministering in step (c) further comprises administering an effectiveamount of a caspase 1, GSDMD, IL-1R IL-1β, IL-6, IL-18, NLRP3,pan-caspase or TNF-α inhibitor.

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Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method for treating a patient infected withSARS-CoV-2 or a variant thereof, wherein the SARS-CoV-2 and the variantthereof have pyroptotic activity, said comprising (a) determining if thepatient is infected with SARS-CoV-2 or the variant thereof, (b)optionally determining if the patient has pyroptotic activity or acomorbidity associated with upregulation of aninflammasome/caspase/pyroptosis axis, and (c) administering to thepatient infected with SARS-CoV-2 or a variant thereof an effectiveamount of about 10 mg/kg to about 100 mg/kg of a compound having theformula

or a derivative, or a prodrug, or a salt, of the compound, wherein theadministering comprises administering a composition comprising theeffective amount of the compound, or the derivative of the compound, orthe prodrug of the compound or the salt of the compound and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinstep (b) is performed and comprises screening a marker indicative ofpyroptotic activity, wherein the marker has increased activity ifpyroptotic activity is present.
 3. The method of claim 2, wherein themarker is NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), IL-1receptor antagonist (IL-1RA), interleukin-1(3 (IL-1β), interleukin-18(IL-18), gasdermin D (GSDMD), caspase 1, lymphopenia, a high neutrophilcount, elevated LDH, ferritin or CRP levels.
 4. The method of claim 1,wherein the effective amount is 300 mg to about 900 mg per dose.
 5. Themethod of claim 4, wherein the effective amount is about 600 mg to about900 mg per dose.
 6. The method of claim 1, wherein the effective amountis about 300 mg to about 3600 mg per day.
 7. The method of claim 4,wherein the 300 mg to about 900 mg per dose is administered three orfour times a day.
 8. The method of claim 7, wherein the 300 mg to about900 mg per dose is administered about every six to about eight hourswithin the day.
 9. The method of claim 1, wherein the administering instep (c) is orally.
 10. The method of claim 2, wherein the administeringin step (c) is orally.
 11. The method of claim 3, wherein theadministering in step (c) is orally.
 12. The method of claim 4, whereinthe administering in step (c) is orally.
 13. The method of claim 5,wherein the administering in step (c) is orally.
 14. The method of claim6, wherein the administering in step (c) is orally.
 15. The method ofclaim 7, wherein the administering in step (c) is orally.
 16. The methodof claim 8, wherein the administering in step (c) is orally.
 17. Themethod of claim 9, wherein the composition is a tablet.
 18. The methodof claim 10, wherein the composition is a tablet.
 19. The method ofclaim 11, wherein the composition is a tablet.
 20. The method of claim12, wherein the composition is a tablet.
 21. The method of claim 13,wherein the composition is a tablet.
 22. The method of claim 14, whereinthe composition is a tablet.
 23. The method of claim 15, wherein thecomposition is a tablet.
 24. The method of claim 16, wherein thecomposition is a tablet.
 25. The method of claim 17, wherein the tabletcomprises a 300 mg dose of the compound, or the derivative of thecompound, or the prodrug of the compound or the salt of the compound.26. The method of claim 1, wherein the administering comprisesadministering the compound or a salt thereof.
 27. The method of claim 9,wherein the administering comprises administering the compound or a saltthereof.
 28. The method of claim 17, wherein the administering comprisesadministering the compound or a salt thereof.
 29. The method of claim25, wherein the administering comprises administering one tablet, twotablets or three tablets.
 30. The method of claim 1, wherein theadministering in step (c) further comprises administering an effectiveamount of a caspase 1, GSDMD, IL-1R, IL-1β, IL-6, IL-18, NLRP3,pan-caspase, TNF-α inhibitor, or an antiviral medication.